Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis

ABSTRACT

A microchip laboratory system and method provide fluid manipulations for a variety of applications, including sample injection for microchip chemical separations. The microchip is fabricated using standard photolithographic procedures and chemical wet etching, with the substrate and cover plate joined using direct bonding. Capillary electrophoresis and electrochromatography are performed in channels formed in the substrate. Analytes are loaded into a four-way intersection of channels by electrokinetically pumping the analyte through the intersection, followed by switching of the potentials to force an analyte plug into the separation channel.

[0001] This invention was made with Government support under contractDE-AC05-84OR21400 awarded by the U.S. Department of Energy to MartinMarietta Energy Systems, Inc. and the Government has certain rights inthis invention.

FIELD OF THE INVENTION

[0002] The present invention relates generally to miniatureinstrumentation for chemical analysis, chemical sensing and synthesisand, more specifically, to electrically controlled manipulations offluids in micromachined channels. These manipulations can be used in avariety of applications, including the electrically controlledmanipulation of fluid for capillary electrophoresis, liquidchromatography, flow injection analysis, and chemical reaction andsynthesis.

BACKGROUND OF THE INVENTION

[0003] Laboratory analysis is a cumbersome process. Acquisition ofchemical and biochemical information requires expensive equipment,specialized labs and highly trained personnel. For this reason,laboratory testing is done in only a fraction of circumstances whereacquisition of chemical information would be useful. A large proportionof testing in both research and clinical situations is done with crudemanual methods that are characterized by high labor costs, high reagentconsumption, long turnaround times, relative imprecision and poorreproducibility. The practice of techniques such as electrophoresis thatare in widespread use in biology and medical laboratories have notchanged significantly in thirty years.

[0004] Operations that are performed in typical laboratory processesinclude specimen preparation, chemical/biochemical conversions, samplefractionation, signal detection and data processing. To accomplish thesetasks, liquids are often measured and dispensed with volumetricaccuracy, mixed together, and subjected to one or several differentphysical or chemical environments that accomplish conversion orfractionation. In research, diagnostic, or development situations, theseoperations are carried out on a macroscopic scale using fluid volumes inthe range of a few microliters to several liters at a time. Individualoperations are performed in series, often using different specializedequipment and instruments for separate steps in the process.Complications, difficulty and expense are often the result of operationsinvolving multiple laboratory processing steps.

[0005] Many workers have attempted to solve these problems by creatingintegrated laboratory systems. Conventional robotic devices have beenadapted to perform pipetting, specimen handling, solution mixing, aswell as some fractionation and detection operations. However, thesedevices are highly complicated, very expensive and their operationrequires so much training that their use has been restricted to arelatively small number of research and development programs. Moresuccessful have been automated clinical diagnostic systems for rapidlyand inexpensively performing a small number of applications such asclinical chemistry tests for blood levels of glucose, electrolytes andgases. Unfortunately due to their complexity, large size and great cost,such equipment, is limited in its application to a small number ofdiagnostic circumstances.

[0006] The desirability of exploiting the advantages of integratedsystems in a broader context of laboratory applications has led toproposals that such systems be miniaturized. In the 1980's, considerableresearch and development effort was put into an exploration of theconcept of biosensors with the hope they might fill the need. Suchdevices make use of selective chemical systems or biomolecules that arecoupled to new methods of detection such as electrochemistry and opticsto transduce chemical signals to electrical ones that can be interpretedby computers and other signal processing units. Unfortunately,biosensors have been a commercial disappointment. Fewer than 20commercialized products were available in 1993, accounting for revenuesin the U.S. of less than $100 million. Most observers agree that thisfailure is primarily technological rather than reflecting amisinterpretation of market potential. In fact, many situations such asmassive screening for new drugs, highly parallel genetic research andtesting, microchemistry to minimize costly reagent consumption and wastegeneration, and bedside or doctor's office diagnostics would greatlybenefit from miniature integrated laboratory systems.

[0007] In the early 1990's, people began to discuss the possibility ofcreating miniature versions of conventional technology. Andreas Manz wasone of the first to articulate the idea in the scientific press. Callingthem “miniaturized total analysis systems,” or “μ-TAS,” he predictedthat it would be possible to integrate into single units microscopicversions of the various elements necessary to process chemical orbiochemical samples, thereby achieving automated experimentation. Sincethat tine, miniature components have appeared, particularly molecularseparation methods and microvalves. However, attempts to combine thesesystems into completely integrated systems have not met with success.This is primarily because precis manipulation of tiny fluid volumes inextremely narrow channels has proven to be a difficult technologicalhurdle.

[0008] One prominent field susceptible to miniaturization is capillaryelectrophoresis. Capillary electrophoresis has become a populartechnique for separating charged molecular species in solution. Thetechnique is performed in small capillary tubes to reduce bandbroadening effects due to thermal convection and hence improve resolvingpower. The small tubes imply that minute volumes of materials, on theorder of nanoliters, must be handled to inject the sample into theseparation capillary tube.

[0009] Current techniques for injection include electromigration andsiphoning of sample from a container into a continuous separation tube.Both of these techniques suffer from relatively poor reproducibility,and electromigration additionally suffers from electrophoreticmobility-based bias. For both sampling techniques the input end of theanalysis capillary tube must be transferred from a buffer reservoir to areservoir holding the sample. Thus, a mechanical manipulation isinvolved. For the siphoning injection, the sample reservoir is raisedabove the buffer reservoir holding the exit end of the capillary for afixed length of time.

[0010] An electromigration injection is effected by applying anappropriately polarized electrical potential across the capillary tubefor a given duration while the entrance end of the capillary is in thesample reservoir. This can lead to sampling bias because adisproportionately larger quantity of the species with higherelectrophoretic mobilities migrate into the tube. The capillary isremoved from the sample reservoir and replaced into the entrance bufferreservoir after the injection duration for both techniques.

[0011] A continuing need exists for methods and apparatuses which leadto improved electrophoretic resolution and improved injection stability.

SUMMARY OF THE INVENTION

[0012] The present invention provides microchip laboratory systems andmethods that allow complex biochemical and chemical procedures to beconducted on a microchip under electronic control. The microchiplaboratory systems comprises a material handling apparatus thattransports materials through a system of interconnected, integratedchannels on a microchip. The movement of the materials is preciselydirected by controlling the electric fields produced in the integratedchannels. The precise control of the movement of such materials enablesprecise mixing, separation, and reaction as needed to implement adesired biochemical or chemical procedure.

[0013] The microchip laboratory system of the present invention analyzesand/or synthesizes chemical materials in a precise and reproduciblemanner. The system includes a body having integrated channels connectinga plurality of reservoirs that store the chemical materials used in thechemical analysis or synthesis performed by the system. In one aspect,at least five of the reservoirs simultaneously have a controlledelectrical potential, such that material from at least one of thereservoirs is transported through the channels toward at least one ofthe other reservoirs. The transportation of the material through thechannels provides exposure to one or more selected chemical or physicalenvironments, thereby resulting in the synthesis or analysis of thechemical material.

[0014] The microchip laboratory system preferably also includes one ormore intersections of integrated channels connecting three or more ofthe reservoirs. The laboratory system controls the electric fieldsproduced in the channels in a manner that controls which materials inthe reservoirs are transported through the intersection(s). In oneembodiment, the microchip laboratory system acts as a mixer or diluterthat combines materials in the intersection(s) by producing anelectrical potential in the intersection that is less than theelectrical potential at each of the two reservoirs from which thematerials to be mixed originate. Alternatively, the laboratory systemcan act as a dispenser that electrokinetically injects precise,controlled amounts of material through the intersection(s).

[0015] By simultaneously applying an electrical potential at each of atleast five reservoirs, the microchip laboratory system can act as acomplete system for performing an entire chemical analysis or synthesis.The five or more reservoirs can be configured in a manner that enablesthe electrokinetic separation of a sample to be analyzed (“the analyte”)which is then mixed with a reagent from a reagent reservoir.Alternatively, a chemical reaction of an analyte and a solvent can beperformed first, and then the material resulting from the reaction canbe electrokinetically separated. As such, the use of five or morereservoirs provides an integrated laboratory system that can performvirtually any chemical analysis or synthesis.

[0016] In yet another aspect of the invention, the microchip laboratorysystem includes a double intersection formed by channels interconnectingat least six reservoirs. The first intersection can be used to inject aprecisely sized analyte plug into a separation channel toward a wastereservoir. The electrical potential at the second intersection can beselected in a manner that provides additional control over the size ofthe analyte plug. In addition, the electrical potentials can becontrolled in a mariner that transports materials from the fifth andsixth reservoirs through the second intersection toward the firstintersection and toward the fourth reservoir after a selected volume ofmaterial from the first intersection is transported through the secondintersection toward the fourth reservoir. Such control can be used topush the analyte plug further down the separation channel while enablinga second analyte plug to be injected through die first intersection.

[0017] In another aspect, the microchip laboratory system acts as amicrochip flow control system to control the flow of material through anintersection formed by integrated channels connecting at least fourreservoirs. The microchip flow control system simultaneously applies acontrolled electrical potential to at least three of the reservoirs suchthat the volume of material transported from the first reservoir to asecond reservoir through the intersection is selectively controlledsolely by the movement of a material from a third reservoir through theintersection. Preferably, the material moved through the third reservoirto selectively control the material transported from the first reservoiris directed toward the same second reservoir as the material from thefirst reservoir. As such, the microchip flow control system acts as avalve or a gate that selectively controls the volume of materialtransported through the intersection. The microchip flow control systemcan also be configured to act as a dispenser that prevents the firstmaterial from moving through the intersection toward the secondreservoir after a selected volume of the first material has passedthrough the intersection. Alternatively, the microchip flow controlsystem can be configured to act as a diluter that mixes the first andsecond materials in the intersection in a manner that simultaneouslytransports the first and second materials from the intersection towardthe second reservoir.

[0018] Other objects, advantages and salient features of the inventionwill become apparent from the following detailed description, whichtaken in conjunction with the annexed drawings, discloses preferredembodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a schematic view of a preferred embodiment of thepresent invention;

[0020]FIG. 2 is an enlarged, vertical sectional view of a channel shown;

[0021]FIG. 3 is a schematic, top view of a microchip according to asecond preferred embodiment of the present invention;

[0022]FIG. 4 is an enlarged view of the intersection region of FIG. 3;

[0023]FIG. 5 are CCD images of a plug of analyte moving through theintersection of the FIG. 30 embodiment;

[0024]FIG. 6 is a schematic top view of a microchip laboratory systemaccording to a third preferred embodiment of a microchip according tothe present invention;

[0025]FIG. 7 is a CCD image of “sample loading mode for rhodamine B”(shaded area);

[0026]FIG. 8(a) is a schematic view of the intersection area of themicrochip of FIG. 6, prior to analyte injection;

[0027]FIG. 8(b) is a CCD fluorescence image taken of the same areadepicted in FIG. 8(a), after sample loading in the pinched mode,

[0028]FIG. 8(c) is a photomicrograph taken of the same area depicted in

[0029]FIG. 8(a), after sample loading in the floating mode;

[0030]FIG. 9 shows integrated fluorescence signals for injected volumeplotted versus time for pinched and floating injections;

[0031]FIG. 10 is a schematic, top view of a microchip according to afourth preferred embodiment of the present invention;

[0032]FIG. 11 is an enlarged view of the intersection region of FIG. 10;

[0033]FIG. 12 is a schematic top view of a microchip laboratory systemaccording to a fifth preferred embodiment according to the presentinvention;

[0034]FIG. 13(a) is a schematic view of a CCD camera view of theintersection area of the microchip laboratory system of FIG. 12;

[0035]FIG. 13(b) is a CCD fluorescence image taken of the same areadepicted in FIG. 13(a), after sample loading in the pinched mode;

[0036] FIGS. 13(c)-13(c) are CCD fluorescence images taken of the samearea depicted in FIG. 13(a), sequentially showing a plug of analytemoving away from the channel intersection at 1, 2, and 3 seconds,respectively, after switching to the run mode;

[0037]FIG. 14 shows two injection profiles for didansyl-lysine injectedfor 2 s with γ equal to 0.97 and 9.7;

[0038]FIG. 15 are electropherograms taken at (a) 3.3 cm, (b) 9.9 cm, and(c) 16.5 cm from the point of injection for rhodamine B (less retained)and sulforhodamine (more retained);

[0039]FIG. 16 is a plot of the efficiency data generated from theelectropherograms of FIG. 15, showing variation of the plate number withchannel length for rhodamine B (square with plus) and sulforhodamine(square with plus) and sulforhodamine (square with dot) with best linearfit (solid lines) for each analyte;

[0040]FIG. 17(a) is an electropherogram of rhodamine B and fluoresceinwith a separation field strength of 1.5 kV/cm and a separation length of0.9 mm;

[0041]FIG. 17(b) is an electropherogram of rhodamine B and fluoresceinwith a separation field strength of 1.5 kV/cm and a separation length of1.6 mm;

[0042]FIG. 17(c) is an electropherogram of rhodamine B and fluoresceinwith a separation field strength of 1.5 kV/cm and a separation length of11.1 mm;

[0043]FIG. 18 is a graph showing variation of the number of plates perunit time as a function of the electric field strength for rhodamine Bat separation lengths of 1.6 mm (circle) and 11.1 mm (square) and forfluorescein at separation lengths of 1.6 mm (diamond) and 11.1 mm(triangle),

[0044]FIG. 19 shows a chromatogram of coumarins analyzed byelectrochromatography using the system of FIG. 12;

[0045]FIG. 20 shows a chromatogram of coumarins resulting from micellarelectrokinetic capillary chromatography using the system of FIG. 12;

[0046] FIGS. 21(a) and 21(b) show the separation of three metal ionsusing the system of FIG. 12;

[0047]FIG. 22 is a schematic, top plan view of a microchip according tothe FIG. 3 embodiment, additionally including a reagent reservoir andreaction channel;

[0048]FIG. 23 is a schematic view of the embodiment of FIG. 20, showingapplied voltages;

[0049]FIG. 24 shows two electropherograms produced using the FIG. 22embodiment;

[0050]FIG. 25 is a schematic view of a microchip laboratory systemaccording to a sixth preferred embodiment of the present invention;

[0051]FIG. 26 shows the reproducibility of the amount injected forarginine and glycine using the system of FIG. 25;

[0052]FIG. 27 shows the overlay of three electrophoretic separationsusing the system of FIG. 25;

[0053]FIG. 28 shows a plot of amounts injected versus reaction timeusing the system of FIG. 25;

[0054]FIG. 29 shows an electropherogram of restriction fragmentsproduced using the system of FIG. 25;

[0055]FIG. 30 is a schematic view of a microchip laboratory systemaccording to a seventh preferred embodiment of the present invention.

[0056]FIG. 31 is a schematic view of the apparatus of FIG. 21, showingsequential applications of voltages to effect desired fluidicmanipulations; and

[0057]FIG. 32 is a graph showing the different voltages applied toeffect the fluidic manipulations of FIG. 23.

DETAILED DESCRIPTION OF THE INVENTION

[0058] Integrated, micro-laboratory systems for analyzing orsynthesizing chemicals require a precise way of manipulating fluids andfluid-bome material and subjecting the fluids to selected chemical orphysical environments that produce desired conversions or partitioning.Given the concentration of analytes that produces chemical conversion inreasonable time scales, the nature of molecular detection, diffusiontimes and manufacturing methods for creating devices on a microscopicscale, miniature integrated micro-laboratory systems lend themselves tochannels having dimensions on the order of 1 to 100 micrometers indiameter. Within this context, electrokinetic pumping has proven to beversatile and effective in transporting materials in microfabricatedlaboratory systems.

[0059] The present invention provides the tools necessary to make use ofelectrokinetic pumping not only in separations, but also to performliquid handling that accomplishes other important sample processingsteps, such as chemical conversions or sample partitioning. Bysimultaneously controlling voltage at a plurality of ports connected bychannels in a microchip structure, it is possible to measure anddispense fluids with great precision mix reagents, incubate reactioncomponents, direct the components towards sites of physical orbiochemical partition and subject the components to detector systems. Bycombining these capabilities on a single microchip, one is able tocreate complete, miniature, integrated automated laboratory systems foranalyzing or synthesizing chemicals.

[0060] Such integrated micro-laboratory systems can be made up ofseveral component elements. Component elements can include liquiddispersing systems, liquid mixing systems, molecular partition systems,detector sights, etc. For example, as described herein, one canconstruct a relatively complete system for the identification ofrestriction endonuclease sites in a DNA molecule. This singlemicrofabricated device thus includes in a single system the functionsthat are traditionally performed by a technician employing pipettors,incubators, gel electrophoresis systems, and data acquisition systems.In this system, DNA is mixed with an enzyme, the mixture is incubated,and a selected volume of the reaction mixture is dispensed into aseparation channel. Electrophoresis is conducted concurrent withfluorescent labeling of the DNA.

[0061] Shown in FIG. 1 is an example of a microchip laboratory system 10configured to implement an entire chemical analysis or synthesis. Thelaboratory system 10 includes six reservoirs 12, 14, 16, 18, 20, and 22connected to cash other by a system of channels 24 micromachined into asubstrate or base member (not shown in FIG. 1), as discussed in moredetail below. Each reservoir 12-22 is in fluid communication with acorresponding channel 26, 28, 30, 32, 34, 36, and 38 of the channelsystem 24. The first channel 26 leading from the first reservoir 12 isconnected to the second channel 28 leading from the second reservoir 14at a first intersection 38. Likewise, the third channel 30 from thethird reservoir 16 is connected to the fourth channel 32 at a secondintersection 40. The first intersection 38 is connected to the secondintersection 40 by a reaction chamber or channel 42. The fifth channel34 from the fifth reservoir 20 is also connected to the secondintersection 40 such that the second intersection 40 is a four-wayintersection of channels 30, 32, 34, and 42. The fifth channel 34 alsointersects the sixth channel 36 from the sixth reservoir 22 at a thirdintersection 44

[0062] The materials stored in the reservoirs preferably are transportedelectrokinetically through the channel system 24 in order to implementthe desire analysis or synthesis. To provide such electrokinetictransport, the laboratory system 10 includes a voltage controller 46capable of applying selectable voltage levels, including ground. Such avoltage controller can be implemented using multiple voltage dividersand multiple relays to obtain the selectable voltage level. The voltagecontroller is connected to an electrode positioned in each of the sixreservoirs 12-22 by voltage lines V1-V6 in order to apply the desiredvoltages to the materials in the reservoirs. Preferably, the voltagecontroller also includes sensor channels S1, S2, and S3 connected to thefirst, second, and third intersections 38, 40, 44, respectively, inorder to sense the voltages present at those intersections.

[0063] The use of electrokinetic transport on microminiaturized planarliquid phase separation devices, described above, is a viable approachfor sample manipulation and as a pumping mechanism for liquidchromatography. The present invention also entails the use ofelectroosmotic flow to mix various fluids in a controlled andreproducible fashion. When an appropriate fluid is placed in a tube madeof a correspondingly appropriate material, functional groups at thesurface of the tube can ionize. In the case of tubing materials that areterminated in hydroxyl groups, protons will leave the surface and enteran aqueous solvent. Under such conditions the surface will have a netnegative charge and the solvent will have an excess of positive charges,mostly in the charged double layer at the surface. With the applicationof an electric field across the tube, the excess cations in solutionwill be attracted to the cathode, or negative electrode. The movement ofthese positive charges through the tube will drag the solvent with them.The steady state velocity is given by equation 1, $\begin{matrix}{v = \frac{{ɛ\xi}\quad E}{4\quad \pi \quad \eta}} & (1)\end{matrix}$

[0064] where v is the solvent velocity, ε is the dielectric constant ofthe fluid, ξ is the zeta potential of the surface, E is the electricfield strength, and π is the solvent viscosity. From equation 1 it isobvious that the fluid flow velocity or flow rate can be controlledthrough the electric field strength. Thus, electroosmosis can be used asa programmable pumping mechanism.

[0065] The laboratory microchip system 10 shown in FIG. 1 could be usedfor performing numerous types of laboratory analysis or synthesis, suchas DNA sequencing or analysis, electrochromatography, micellarelectrokinetic capillary chromatography (MECC), inorganic ion analysis,and gradient elution liquid chromatography, as discussed in more detailbelow. The fifth channel 34 typically is used for electrophoretic orelectrochromatographic separations and thus may be referred to incertain embodiments as a separation channel or column. The reactionchamber 42 can be used to mix any two chemicals stored in the first andsecond reservoirs 12, 14. For example, DNA from the first reservoir 12could be mixed with an enzyme from the second reservoir 14 in the firstintersection 38 and the mixture could be incubated in the reactionchamber 42. The incubated mixture could then be transported through thesecond intersection 40 into the separation column 34 for separation. Thesixth reservoir 22 can be used to store a fluorescent label that ismixed in the third intersection 44 with the materials separated in theseparation column 34. An appropriate detector (D) could then be employedto analyze the labeled materials between the third intersection 44 andthe fifth reservoir 20. By providing for a pre-separation columnreaction in the first intersection 38 and reaction chamber 42 and apost-separation column reaction in the third intersection 44, thelaboratory system 10 can be used to implement many standard laboratorytechniques normally implemented manually in a conventional laboratory.In addition, the elements of the laboratory system 10 could be used tobuild a more complex system to solve more complex laboratory procedures.

[0066] The laboratory microchip system 10 includes a substrate or basemember (not shown in FIG. 1) which can be an approximately two inch byone inch piece of microscope slide (Corning, Inc. #2947). While glass isa preferred material, other similar materials may be used, such as fusedsilica, crystalline quartz, fused quartz, plastics, and silicon (if thesurface is treated sufficiently to alter its resistivity). Preferably, anon-conductive material such as glass or fused quartz is used to allowrelatively high electric fields to be applied to electrokineticallytransport materials through channels in the microchip. Semiconductingmaterials such as silicon could also be used, but the electric fieldapplied would normally need to be kept to a minimum (approximately lessthan 300 volts per centimeter using present techniques of providinginsulating layers), which may provide insufficient electrokineticmovement.

[0067] The channel pattern 24 is formed in a planar surface of thesubstrate using standard photolithographic procedures followed bychemical wet etching. The channel pattern may be transferred onto thesubstrate with a positive photoresist (Shipley 1811) and an e-beamwritten chrome mask (Institute of Advanced Manufacturing Sciences,Inc.). The pattern may be chemically etched using HF/NH₄F solution

[0068] After forming the channel pattern, a cover plate may then bebonded to the substrate using a direct bonding technique whereby thesubstrate and the cover plate surfaces are first hydrolyzed in a diluteNH₄OH₄H₂O₂ solution and then joined. The assembly is then annealed atabout 500° C. in order to insure proper adhesion of the cover plate tothe substrate.

[0069] Following bonding of the cover plate, the reservoirs are affixedto the substrate, with portions of the cover plate sandwichedtherebetween, using epoxy or other suitable means. The reservoirs can becylindrical with open opposite axial ends. Typically, electrical contactis made by placing a platinum wire electrode in each reservoirs. Theelectrodes are connected to a voltage controller 46 which applies adesired potential to select electrodes, in a manner described in moredetail below.

[0070] A cross section of the first channel is shown in FIG. 2 and isidentical to the cross section of each of the other integrated channels.When using a non-crystalline material (such as glass) for the substrate,and when the channels are chemically wet etched, an isotropic etchoccurs, i.e., the glass etches uniformly in all directions, and theresulting channel geometry is trapezoidal. The trapezoidal cross sectionis due to “undercutting” by the chemical etching process at the edge ofthe photoresist. In one embodiment, the channel cross section of theillustrated embodiment has dimensions of 5.2 μm in depth, 57 μm in widthat the top and 45 μm in width at the bottom. In another embodiment, thechannel has a depth “d” of 10 μm, an upper width “w1” of 90 μm, and alower width “w2” of 70 μm.

[0071] An important aspect of the present invention is the controlledelectrokinetic transportation of materials through the channel system24. Such controlled electrokinetic transport can be used to dispense aselected amount of material from one of the reservoirs through one ormore intersections of the channel structure 24. Alternatively, as notedabove, selected amounts of materials from two reservoirs can betransported to an intersection where the materials can be mixed indesired concentrations.

[0072] Gated Dispenser

[0073] Shown in FIG. 3 is a laboratory component 10A that can be used toimplement a preferred method of transporting materials through a channelstructure 24A The A following each number in FIG. 3 indicates that itcorresponds to an analogous element of FIG. 1 of the same number withoutthe A. For simplicity, the electrodes and the connections to the voltagecontroller that controls the transport of materials through the channelsystem 24A are not shown in FIG. 3.

[0074] The microchip laboratory system 10A shown in FIG. 3 controls theamount of material from the first reservoir 12A transported through theintersection 40A toward the fourth reservoir 20A by electrokineticallyopening and closing access to the intersection 40A from the firstchannel 26A. As such the laboratory microchip system 10A essentiallyimplements a controlled electrokinetic valve. Such an electrokineticvalve can be used as a dispenser to dispense selected volumes of asingle material or as a mixer to mix selected volumes of pluralmaterials in the intersection 40A. In general, electro-osmosis is usedto transport “fluid materials” and electrophoresis is used to transportions without transporting the fluid material surrounding the ions.Accordingly, as used herein, the term “material” is used broadly tocover any form of material, including fluids and ions.

[0075] The laboratory system 10A provides a continuous unidirectionalflow of fluid through the separation channel 34A. This injection ordispensing scheme only requires that the voltage be changed or removedfrom one (or two) reservoirs and allows the fourth reservoir 20A toremain at ground potential. Ts will allow injection and separation to beperformed with a single polarity power supply.

[0076] An enlarged view of the intersection 40A is shown in FIG. 4. Thedirectional arrows indicate the time sequence of the flow profiles atthe intersection 40A. The solid arrows show the initial flow pattern.Voltages at the various reservoirs are adjusted to obtain the describedflow patterns. The initial flow pattern brings a second material fromthe second reservoir 16A at a sufficient rate such that all of the firstmaterial transported from reservoir 12A to the intersection 40A ispushed toward the third reservoir 18A. In general, the potentialdistribution will be such that the highest potential is in the secondreservoir 16A, a slightly lower potential in the first reservoir 12A,and yet a lower potential in the third reservoir 18A, with the fourthreservoir 20A being grounded. Under these conditions, the flow towardsthe fourth reservoir 20A is solely the second material from the secondreservoir 16A.

[0077] To dispense material from the first reservoir 12A through theintersection 40A, the potential at the second reservoir 16A can beswitched to a value less than the potential of the first reservoir 12Aor the potentials at reservoirs 16A and/or 18A, can be floatedmomentarily to provide the flow shown by the short dashed arrows in FIG.4. Under these conditions, the primary flow will be from the firstreservoir 12A down towards the separation channel waste reservoir 20A.The flow from the second and third reservoirs 16A, 18A will be small andcould be in either direction. This condition is held long enough totransport a desired amount of material from the fir reservoir 12Athrough the intersection 40A and into the separation channel 34A. Aftersufficient time for the desired material to pass through theintersection 40A, the voltage distribution is switched back to theoriginal values to prevent additional material from the first reservoir12A from flowing through the intersection 40A toward the separationchannel 34A.

[0078] One application of such a “gated dispenser” is to inject acontrolled, variable-sized plug of analyte from the first reservoir 12Afor electrophoretic or chromatographic separation in the separationchannel 34A. In such a system, the first reservoir 12A stores analyte,the second reservoir 16A stores an ionic buffer, the third reservoir 18Ais a first waste reservoir and the fourth reservoir 20A is a secondwaste reservoir. To inject a small variable plug of analyte from thefirst reservoir 12A, the potentials at the buffer and first wastereservoirs 16A, 18A are simply floated for a short period of time (≈100ms) to allow the analyte to migrate down the separation column 34A. Tobreak off the injection plug, the potentials at the buffer reservoir 16Aand the first waste reservoir 18A are reapplied. Alternatively, thevalving sequence could be effected by bringing reservoirs 16A and 18A tothe potential of the intersection 40A and then returning them to theiroriginal potentials A shortfall of this method is that the compositionof the injected plug has an electrophoretic mobility bias whereby thefaster migrating compounds are introduced preferentially into theseparation column 34A over slower migrating compounds.

[0079] In FIG. 5, a sequential view of a plug of analyte moving throughthe intersection of the FIG. 3 embodiment can be seen by CCD images Theanalyte being pumped through the laboratory system 10A was rhodamine B(shaded area), and the orientation of the CCD images of the injectioncross or intersection is the same as in FIG. 3. The first image, (A),shows the analyte being pumped through the injection cross orintersection toward the first waste reservoir 18A prior to theinjection. The second image, (B), shows the analyte plug being injectedinto the separation column 34A. The third image, (C), depicts theanalyte plug moving away from the injection intersection after aninjection plug has been completely introduced into the separation column34A. The potentials at the buffer and first waste reservoirs 16A, 18Awere floated for 100 ms while the sample moved into the separationcolumn 34A. By the time of the (C) image, the closed gate mode hasresumed to stop further analyte from moving through the intersection 40Ainto the separation column 34A, and a clean injection plug with a lengthof 142 μm has been introduced into the separation column. As discussedbelow, the gated injector contributes to only a minor fraction of thetotal plate height. The injection plug length (volume) is a function ofthe time of the injection and the electric field strength in the column.The shape of the injected plug is skewed slightly because of thedirectionality of the cleaving buffer flow. However, for a giveninjection period, the reproducibility of the amount injected, determinedby integrating the peak area, is 1% RSD for a series of 10 replicateinjections.

[0080] Electrophoresis experiments were conducted using the microchiplaboratory system 10A of FIG. 3, and employed methodology according tothe present invention. Chip dynamics were analyzed using analytefluorescence. A charge coupled device (CCD) camera was used to monitordesignated areas of the chip and a photomultiplier tube (PMT) trackedsingle point events. The CCD (Princeton Instruments, Inc. TE/CCD-512TKM)camera was mounted on a stereo microscope (Nikon SMZ-U), and thelaboratory system 10A was illuminated using an argon ion laser (514.5nm, Coherent Innova 90) operating at 3 W with the beam expanded to acircular spot ≈2 cm in diameter. The PMT, with collection optics, wassituated below the microchip with the optical axis perpendicular to themicrochip surface. The laser was operated at approximately 20 mW, andthe beam impinged upon the microchip at a 45° angle from the microchipsurface and parallel to the separation channel. The laser beam and PMTobservation axis were separated by a 135° angle. The point detectionscheme employed a helium-neon laser (543 nm, PMS Electro-opticsLIMP-0051) with an electrometer (Keithley 617) to monitor response ofthe PMT (Oriel 77340). The voltage controller 46 (Spellman CZE 1000R)for electrophoresis was operated between 0 and +4.4 kV relative toground.

[0081] The type of gated injector described with respect to FIGS. 3 and4 show electrophoretic mobility based bias as do conventionalelectroosmotic injections. Nonetheless, this approach has simplicity involtage switching requirements and fabrication and provides continuousunidirectional flow through the separation channel. In addition, thegated injector provides a method for valving a variable volume of fluidinto the separation channel 34A in a manner that is precisely controlledby the electrical potentials applied.

[0082] Another application of the gated dispenser 10A is to dilute ormix desired quantities of materials in a controlled manner. To implementsuch a mixing scheme in order to mix the materials from the first andsecond reservoirs 12A, 16A, the potentials. in the first and secondchannels 26A, 30A need to be maintained higher than the potential of theintersection 40A during mixing. Such potentials will cause the materialsfrom the first and second reservoirs 12A and 16A to simultaneously movethrough the intersection 40A and thereby mix the two materials. Thepotentials applied at the first and second reservoirs 12A, 16A can beadjusted as desired to achieve the selected concentration of eachmaterial. After dispensing the desired amounts of each material, thepotential at the second reservoir 16A may be increased in a mannersufficient to prevent further material from the first reservoir 12A frombeing transported through the intersection 40A toward the thirdreservoir 30A.

[0083] Analyte Injector

[0084] Shown in FIG. 6 is a microchip analyte injector 10B according tothe present invention. The channel pattern 24B has four distinctchannels 26B, 30B, 32B, and 34B micromachined into a substrate 49 asdiscussed above. Each channel has an accompanying reservoir mountedabove the terminus of each charnel portion, and all four channelsintersect at one end in a four way intersection 40B. The opposite endsof each section provide termini that extend just beyond the peripheraledge of a cover plate 49′ mounted on the substrate 49. The analyteinjector 10B shown in FIG. 6 is substantially identical to the gateddispenser 10A except that the electrical potentials are applied in amanner that injects a volume of material from reservoir 16% through theintersection 40B rather than from the reservoir 12B and the volume ofmaterial injected is controlled by the size of the intersection.

[0085] The embodiment shown in FIG. 6 can be used for various materialmanipulations. In one application the laboratory system is used toinject an analyte from an analyte reservoir 16B through the intersection40B for separation in the separation channel 34B. The analyte injector10B can be operated in either “load” mode or a “run” mode. Reservoir 16Bis supplied with an analyte and reservoir 12B with buffer. Reservoir 18Bacts as an analyte waste reservoir, and reservoir 20B acts as a wastereservoir.

[0086] In the “load” mode, at least two types of analyte introductionare possible. In the first, known as a “floating” loading, a potentialis applied to the analyte reservoir 16B with reservoir 18B grounded. Atthe same time, reservoirs 12B and 20B are floating, meaning that theyare neither coupled to the power source, nor grounded.

[0087] The second load mode is “pinched” loading mode, whereinpotentials are simultaneously applied at reservoirs 12B, 16B, and 20B,with reservoir 18B grounded in order to control the injection plug shapeas discussed in more detail below. As used herein, simultaneouslycontrolling electrical potentials at plural reservoirs means that theelectrodes are connected to a operating power source at the samechemically significant time period. Floating a reservoir meansdisconnecting the electrode in the reservoir font the power source andthus the electrical potential at the reservoir is not controlled.

[0088] In the “run” mode, a potential is applied to the buffer reservoir12B with reservoir 20B grounded and with reservoirs 16B and 18B atapproximately half of the potential of reservoir 12B. During the runmode, the relatively high potential applied to the buffer reservoir 12Bcauses the analyte in the intersection 40B to move toward the wastereservoir 20B in the separation column 34B.

[0089] Diagnostic experiments were performed using rhodamine B andsulforhodamine 101 (Exciton Chemical Co., Inc.) as the analyte at 60 μMfor the CCD images and 6 μM for the point detection. A sodiumtetraborate buffer (50 mM, pH 9.2) was the mobile phase in theexperiments. An injection of spatially well defined small volume (≈100μL) and of small longitudinal extent (≈100 μm), injection is beneficialwhen performing these types of analyses.

[0090] The analyte is loaded into the injection cross as a frontalelectropherogram, and once the front of the slowest analyte componentpasses through the injection cross or intersection 40B, the analyte isready to be analyzed. In FIG. 7, a CCD image (the area of which isdenoted by the broken line square) displays the flow pattern of theanalyte 54 (shaded area) and the buffer (white area) through the regionof the injection intersection 40B.

[0091] By pinching the flow of the analyte, the volume of the analyteplug is stable over time. The slight asymmetry of the plug shape is dueto the different electric field strengths in the buffer channel 26B (470V/Cm) and the separation channel 34B (100 V/cm) when 1.0 kV is appliedto the buffer, the analyte and the waste reservoirs, and the analytewaste reservoir is grounded. However, the different field strengths donot influence the stability of the analyte plug injected. Ideally, whenthe analyte plug is injected into the separation channel 34B, only theanalyze in the injection cross or intersection 40B would migrate intothe separation channel.

[0092] The volume of the injection plug in the injection cross isapproximately 120 pL with a plug length of 130 μm. A portion of theanalyte 54 in the analyte channel 30B and the analyte waste channel 32Bis drawn into the separation channel 34B. Following the switch to theseparation (run) mode, the volume of the injection plug is approximately250 pL with a plug length of 208 μm. These dimensions are estimated froma series of CCD images taken immediately after the switch is made to theseparation mode.

[0093] The two modes of loading were tested for the analyte introductioninto the separation channel 34B. The analyte was placed in the analytereservoir 16B, and in both injection schemes was “transported” in thedirection of reservoir 18B, a waste reservoir. CCD images of the twotypes of injections are depicted in FIGS. 8(a)-8(c). FIG. 8(a)schematically shows the intersection 40B, as well as the end portions ofchannels.

[0094] The CCD image of FIG. 8(b) is of loading in the pinched mode,just prior to being switched to the run mode. In the pinched mode,analyte (shown as white against the dark background) is pumpedelectrophoretically and electroosmotically from reservoir 16B toreservoir 18B (left to right) with buffer from the buffer reservoir 12B(top) and the waste reservoir 20B (bottom) traveling toward reservoir18B (right). The voltages applied to reservoirs 12B, 16B, 18B, and 20Bwere 90%, 90%, 0, and 100% respectively, of the power supply outputwhich correspond to electric field strengths in the correspondingchannels of 400, 270, 690 and 20 V/cm, respectively. Although thevoltage applied to the waste reservoir 20B is higher than voltageapplied to the analyte reservoir 18B, the additional length of theseparation channel 34B compared to the analyte channel 30B providesadditional electrical resistance, and thus the flow from the analytebuffer 16B into the intersection predominates. Consequently, the analytein the injection cross or intersection 40B has a trapezoidal shape andis spatially constricted in the channel 32B by this material transportpattern

[0095]FIG. 8(c) shows a floating mode loading. The analyte is pumpedfrom reservoir 16B to 18B as in the pinched injection except nopotential is applied to reservoirs 12B and 20B. By not controlling theflow of mobile phase (buffer) in channel portions 26B and 34B, theanalyte is free to expand into these channels through convective anddiffusive flow, thereby resulting in an extended injection plug.

[0096] When comparing the pinched and floating injections, the pinchedinjection is superior in three areas: temporal stability of the injectedvolume, the precision of the injected volume, and plug length. When twoor more analytes with vastly different mobilities are to be analyzed, aninjection with temporal stability insures that equal volumes of thefaster and slower moving analytes are introduced into the separationcolumn or channel 34B. The high reproducibility of the injection volumefacilitates the ability to perform quantitative analysis. A smaller pluglength leads to a higher separation efficiency and, consequently, to agreater component capacity for a given instrument and to higher speedseparations.

[0097] To determine the temporal stability of each mode., a series ofCCD fluorescence images were collected at 1.5 second intervals startingjust prior to the analyte reaching the injection intersection 40B. Anestimate of the amount of analyte that is injected was determined byintegrating the fluorescence in the intersection 40B and channels 26Band 34B. This fluorescence is plotted versus time in FIG. 9.

[0098] For the pinched injection, the injected volume stabilizes in afew seconds and has a stability of 1% relative standard deviation (RSD),which is comparable to the stability of the illuminating laser. For thefloating injection, the amount of analyte to be injected into theseparation channel 34B increases with time because of the dispersiveflow of analyte into channels 26B and 34B. For a 30 second injection,the volume of the injection plug is ca. 90 pL and stable for the pinchedinjection versus ca. 300 pL and continuously increasing with time for afloating injection.

[0099] By monitoring the separation channel at a point 0.9 cm from theintersection 40B, the reproducibility for the pinched injection it odewas tested by integrating the area of the band profile followingintroduction into the separation channel 34B. For six injections with aduration of 40 seconds, the reproducibility for the pinched injection is0.7% RSD. Most of this measured instability is from the opticalmeasurement system. The pinched injection has a higher reproducibilitybecause of the temporal stability of the volume injected. Withelectronically controlled voltage switching, the RSD is expected toimprove for both schemes.

[0100] The injection plug width and, ultimately, the resolution betweenanalytes depends largely on both the flow pattern of the analyte and thedimensions of the injection cross or intersection 40B. For this column,the width of the channel at the top is 90 μm but a channel width of 10μm is feasible which would lead to a decrease in the volume of theinjection plug from 90 pL down to 1 pL with a pinched injection.

[0101] There are situations where it may not be desirable to reverse theflow in the separation channel as described above for the “pinched” and“floating” injection schemes. Examples of such cases might be theinjection of a new sample plug before the preceding plug has beencompletely eluted or the use of a post-column reactor where reagent iscontinuously being injected into the end of the separation column. Inthe latter case, it would in general not be desirable to have thereagent flowing back up into the separation channel.

[0102] Alternate Analyte Injector

[0103]FIG. 10 illustrates an alternate analyte injector system 10Chaving six different ports or channels 26C, 30C, 32C, 34C, 56, and 58respectively connected to six different reservoirs 12C, 16C, 11C, 20C,60, and 62. The letter C after each element number indicates that theindicated element is analogous to a correspondingly numbered elements ofFIG. 1. The microchip laboratory system 10C is similar to laboratorysystems 10, 10A, and 10B described previously, in that an injectioncross or intersection 40C is provided. In the FIG. 10 embodiment, asecond intersection 64 and two additional reservoirs 60 and 62 are alsoprovided to overcome the problems with reversing the flow in theseparation channel.

[0104] Like the previous embodiments, the analyte injector system 10Ccan be used to implement an analyte separation by electrophoresis orchromatography or dispense material into some other processing element.In the laboratory system 10C, the reservoir 12C contains separatingbuffer, reservoir 16C contains the analyte, and reservoirs 18C and 20Care waste reservoirs. Intersection 40C preferably is operated in thepinched mode as in the embodiment shown in FIG. 6. The lowerintersection 64, in fluid communication with reservoirs 60 and 62, areused to provide additional flow so that a continuous buffer stream canbe directed down towards the waste reservoir 20C and, when needed,upwards toward the injection intersection 40C. Reservoir 60 and attachedchannel 56 are not necessary, although they improve performance byreducing band broadening as a plug passes the lower intersection 64. Inmany cases, the flow from reservoir 60 will be symmetric with that fromreservoir 62.

[0105]FIG. 11 is an enlarged view of the two intersections 40C and 64.The different types of arrows show the flow directions at giveninstances in time for injection of a plug of analyte into the separationchannel. The solid arrows show the initial flow pattern where theanalyte is electrokinetically pumped into the upper intersection 40C and“pinched” by material flow from reservoirs 12C, 60, and 62 toward thissame intersection. Flow away from the injection intersection 40C iscarried to the analyte waste reservoir 18C. The analyte is also flowingfrom the reservoir 16C to the analyte waste reservoir 18C. Under theseconditions, flow from reservoir 60 (and reservoir 62) is also going downthe separation channel 34C to the waste reservoir 20C. Such a flowpattern is created by simultaneously controlling the electricalpotentials at all six reservoirs.

[0106] A plug of the analyte is injected through the injectionintersection 40C into the separation channel 34C by switching to theflow profile shown by the short dashed arrows. Buffer flows down fromreservoir 12C to the injection intersection 40C and towards reservoirs16C, 18C, and 20C. This flow profile also pushes the analyte plug towardwaste reservoir 20C into the separation channel 34C is described before.This flow profile is held for a sufficient length of time so as to movethe analyte plug past the lower intersection 64. The flow of buffer fromreservoirs 60 and 62 should be low as indicated by the short arrow andinto the separation channel 34C to minimize distortion.

[0107] The distance between the upper and lower intersections 40C and64, respectively, should be as small as possible to minimize plugdistortion and criticality of timing in the switching between the twoflow conditions. Electrodes for sensing the electrical potential mayalso be placed at the lower intersection and in the channels 56 and 58to assist in adjusting the electrical potentials for proper flowcontrol. Accurate flow control at the lower intersection 64 may benecessary to prove it undesired band broadening.

[0108] After the sample plug passes the lower intersection, thepotentials are switched back to the initial conditions to give theoriginal flow profile as shown with the long dashed arrows. This flowpattern will allow buffer flow into the separation channel 34C while thenext analyte plug is being transported to the plug forming region in theupper intersection 40C. This injection scheme will allow a rapidsuccession of injections to be made and may be very important forsamples that are slow to migrate or if it takes a long time to achieve ahomogeneous sample at the upper intersection 40C such as with entangledpolymer solutions. This implementation of the pinched injection alsomaintains unidirectional flow through the separation channel as might berequired for a post-column reaction as discussed below with respect toFIG. 22.

[0109] Serpentine Channel

[0110] Another embodiment of the invention is the modofied analyteinjector system 10D shown in FIG. 12. The laboratory system 10D shown inFIG. 12 is substantially identical to the laboratory system 10B shown inFIG. 6, except that the separation channel 34D follows a serpentinepath. The serpentine path of the separation channel 34D allows thelength of the separation channel to be greatly increased withoutsubstantially increasing the area of the substrate 49D needed toimplement the serpentine path. Increasing the length of the separationchannel 34D increases the ability of the laboratory system 10D todistinguish elements of an analyte. In one particularly preferredembodiment, the enclosed length (that which is covered by the coverplate 49D′) of the channels extending from reservoir 16D to reservoir18D is 19 mm, while the length of channel portion 26D is 6.4 mm andchannel 34D is 171 mm. The turn radius of each turn of the channel 34D,which serves as a separation column, is 0.16 mm.

[0111] To perform a separation using the modified analyte injectorsystem 10D, an analyte is first loaded into the injection intersection40D using one of the loading methods described above. After the analytehas been loaded into the intersection 40D of the microchip laboratorysystem 10, the voltages are manually switched from the loading mode tothe run (separation) mode of operation. FIGS. 13(a)-13(e) illustrate aseparation of rhodamine B (less retained) and sulforhodamine (moreretained) using the following conditions: E_(inj)=400 V/cm, E_(run)=150V/cm buffer=50 mM sodium tetraborate at pH 9.2. The CCD imagedemonstrate the separation process at 1 second intervals, with FIG.13(a) showing a schematic of the section of the chip imaged, and withFIGS. 13(b)-13(e) showing the separation unfold.

[0112]FIG. 13(b) again shows the pinched injection with the appliedvoltages at reservoirs 12D, 16D, and 20D equal and reservoir 18Dgrounded. FIGS. 13(c)-13(e) shows the plug moving away from theintersection at 1, 2, and 3 seconds, respectively, after switching tothe run mode. In FIG. 13(c), tire injection plug is migrating around a90° turn, and band distortion is visible date to the inner portion ofthe plug traveling less distance than the outer portion. By FIG. 13(d),the analytes have separated into distinct bands, which are distorted inthe shape of a parallelogram. In FIG. 13(c), the bands are wellseparated and have attained a more rectangular shape, collapsing of theparallelogram, due to radial diffusion, an additional contribution toefficiency loss.

[0113] When the switch is made from the load mode to the run mode, aclean break of the injection plug from the analyte stream is desired toavoid tailing. This is achieved by pumping the mobile phase or bufferfrom channel 26D into channels 30D, 32D, and 34D simultaneously bymaintaining the potential at the intersection 40D below the potential ofreservoir 12D and above the potentials of reservoirs 16D, 18D, and 20D.

[0114] In the representative experiments described herein, theintersection 40D was maintained at 66% of the potential of reservoir 12Dduring the run mode. This provided sufficient flow of the analyte backaway from the injection intersection 40D down channels 30D and 32Dwithout decreasing the field strength in the separation channel 34Dsignificantly. Alternate channel designs would allow a greater factionof the potential applied at reservoir 12D to be dropped across theseparation channel 34D, thereby improving efficiency

[0115] This three way flow is demonstrated in FIGS. 13(c)-13(e) as theanalytes in channels 30D and 32D (left and right, respectively) movefurther away from the intersection with time. Three way flow permitswell-defined, reproducible injections with minimal bleed of the analyteinto the separation channel 34D.

[0116] Detectors

[0117] In most applications envisaged for these integrated microsystemsfor chemical analysis or synthesis it will be necessary to quantify thematerial present in a channel at one or more positions similar toconventional laboratory measurement processes. Techniques typicallyutilized for quantification include, but are not limited to, opticalabsorbance, refractive index changes, fluorescence emission,chemiluminescence, various forms of Raman spectroscopy, electricalconductometric measurements, electrochemical amperiometric measurements,acoustic wave propagation measurements.

[0118] Optical absorbence measurements are commonly, employed withconventional laboratory analysis systems because of the generality ofthe phenomenon in the UV portion of the electromagnetic spectrum.Optical absorbence is commonly determined by measuring the attenuationof impinging optical power as it passes through a known length ofmaterial to be quantified. Alternative approaches are possible withlaser technology including photo acoustic and photo thermal techniques.Such measurements can be utilized with the microchip technologydiscussed here with the additional advantage of potentially integratingoptical wave guides on microfabricated devices. The use of solid-stateoptical sources such as LEDs and diode lasers with and without frequencyconversion elements would be attractive for reduction of system size.Integration of solid state optical source and detector technology onto achip does not presently appear viable but may one day be of interest.

[0119] Refractive index detectors have also been commonly used forquantification of flowing stream chemical analysis systems because ofgenerality of the phenomenon but have typically been less sensitive thanoptical absorption. Laser based implementations of refractive indexdetection could provide adequate sensitivity in some situations and haveadvantages of simplicity. Fluorescence emission (or fluorescencedetection) is an extremely sensitive detection technique and is commonlyemployed for the analysis of biological materials. This approach todetection has much relevance to miniature chemical analysis andsynthesis devices because of the sensitivity of the technique and thesmall volumes that can be manipulated and analyzed (volumes in thepicoliter range are feasible). For example, a 100 pL sample volume with1 nM concentration of analyte would have only 60,000 analyte moleculesto be processed and detected. There are several demonstrations in theliterature of detecting a single molecule in solution by fluorescencedetection. A laser source is often used as the excitation source forultrasensitive measurements but conventional light sources such as raregas discharge lamps and light emitting diodes (LEDs) are also used. Thefluorescence emission can be detected by a photomultiplier tube,photodiode or other light sensor. An array detector such as a chargecoupled device (CCD) detector can be used to image an analyte spatialdistribution.

[0120] Raman spectroscopy can be used as a detection method formicrochip devices with the advantage of gaining molecular vibrationalinformation, but with the disadvantage of relatively poor sensitivity.Sensitivity has been increased through surface enhanced Ramanspectroscopy (SERS) effects but only at the research level. Electricalor electrochemical detection approaches are also of particular interestfor implementation on microchip devices due to the ease, of integrationonto a microfabricated structure and the potentially high sensitivitythat can be attained. The most general approach to electricalquantification is a conductometric measurement, i.e., a measurement ofthe conductivity of an ionic sample. The presence of an ionized analytecan correspondingly increase the conductivity of a fluid and thus allowquantification. Amperiometric measurements imply the measurement of thecurrent through an electrode at a given electrical potential due to thereduction or oxidation of a molecule at the electrode. Some selectivitycan be obtained by controlling the potential of the electrode but it isminimal. Amperiometric detection is a less general technique thanconductivity because not all molecules can be reduced or oxidized withinthe limited potentials that can be used with common solvents.Sensitivities in the 1 nM range have been demonstrated in small volumes(10 nL). The other advantage of this technique is that the number ofelectrons measured (through the current) is equal to the number ofmolecules present. The electrodes required for either of these detectionmethods can be included on a microfabricated device through aphotolithographic patterning and metal deposition process. Electrodescould also be used to initiate a chemiluminescence detection process,i.e., an excited state molecule is generated via an oxidation-reductionprocess which then transfers its energy to an analyte molecule,subsequently emitting a photon that is detected.

[0121] Acoustic measurements can also be used for quantification ofmaterials but have not been widely used to date. One method that hasbeen used primarily for gas phase detection is the attenuation or phaseshift of a surface acoustic wave (SAW). Adsorption of material to thesurface of a substrate where a SAW is propagating affects thepropagation characteristics and allows a concentration determination.Selective sorbents on the surface of the SAW device are often used.Similar techniques may be useful in the devices described herein.

[0122] The mixing capabilities of the microchip laboratory systemsdescried herein lend themselves to detection processes that include theaddition of one or more reagents. Derivatization reactions are commonlyused in biochemical assays. For example, amino acids, peptides andproteins are commonly labeled with dansylating reagents oro-phthaldialdehyde to produce fluorescent molecules that are easilydetectable. Alternatively, an enzyme could be used as a labelingmolecule and reagents, including substrate, could be added to provide anenzyme amplified detection scheme, i.e., the enzyme produces adetectable product. These are many examples where such an approach hasbeen used in conventional laboratory procedures to enhance detection,either by absorbence or fluorescence. A third example of a detectionmethod that could benefit from integrated mixing methods ischemiluminescence detection. In these types of detection scenarios, areagent and a catalyst are mixed with an appropriate target molecule toproduce an excited state molecule that emits a detectable photon.

[0123] Analyte Stacking

[0124] To enhance the sensitivity of the microchip laboratory system10D, an analyte pre-concentration can be performed prior to theseparation. Concentration enhancement is a valuable tool especially whenanalyzing environmental samples and biological materials, two areastargeted by microchip technology. Analyte stacking is a convenienttechnique to incorporate with electrophoretic analyses. To employanalyte stacking, the analyte is prepared in a buffer with a lowerconductivity than the separation buffer. The difference in conductivitycauses the ions in the analyte to stack at the beginning or end of theanalyte plug, thereby resulting in a concentrated analyte plug portionthat is detected more easily. More elaborate preconcentration techniquesinclude two and three buffer systems, i.e., transient isotachophoreticpreconcentration. It will be evident that the greater the number ofsolutions involved, the more difficult the injection technique is toimplement. Preconcentration steps are well suited for implementation ona microchip. Electroosmotically driven flow enables separation andsample buffers to be controlled without the use of valves or pumps. Lowdead volume connections between channels can be easily fabricatedenabling fluid manipulation with high precision, speed andreproducibility.

[0125] Referring again to FIG. 12, the pre-concentration of the analyteis performed at the top of the separation channel 34D using a modifiedgated injection to stack the analyte. First, an analyte plug isintroduced onto the separation channel 34D using electroosmotic flow.The analyte plug is then followed by more separation buffer from thebuffer reservoir 16D. At this point, the analyte stacks at theboundaries of the analyte and separation buffers. Dansylated amino acidswere used as the analyte, which are anions that stack at the rearboundary of the analyte buffer plug. Implementation of the analytestacking is described along with the effects of the stacking on both theseparation efficiency and detection limits.

[0126] To employ a gated injection using the microchip laboratory system10D, the analyte is stored in the top reservoir 12D and the buffer isstored in the left reservoir 16D. The gated injection used for theanalyte stacking is performed on an analyte having an ionic strengththat is less than that of the running buffer. Buffer is transported byelectroosmosis from the buffer reservoir 16D towards both the analytewaste and waste reservoirs 18D, 20D. This buffer stream prevents theanalyte from bleeding into the separation channel 34D. Within arepresentative embodiment, the relative potentials at the buffer,analyte, analyte waste and waste reservoirs are 1, 0.9, 0.7 and 0,respectively. For 1 kV applied to the microchip, the field strengths inthe butler, analyte, analyte waste, and separation channels during theseparation are 170, 130, 180, and 120 V/cm, respectively

[0127] To inject the analyte onto the separation channel 34D, thepotential at the buffer reservoir 16D is floated (opening of the highvoltage switch) for a brief period of time (0.1 to 10 s), and analytemigrates into the separation channel. For 1 kV applied to the microchip,the field strengths in the buffer, sample, sample waste, and separationchannels during the injection are 0, 240, 120, and 110 V/cm,respectively. To break off the analyte plug, the potential at the bufferreservoir 16D is reapplied (closing of a high voltage switch). Thevolume of the analyte plug is a function of the injection time, electricfield strength, and electrophoretic mobility

[0128] The separation buffer and analyte compositions can be quitedifferent, yet with the gated injections the integrity of both theanalyte and buffer streams can be alternately maintained in theseparation channel 34D to perform the stacking operation. The analytestacking depends on the relative conductivity of the separation bufferto analyte, γ. For example, with a 5 mM separation buffer and a 0.516 mMsample (0.016 mM dansyl-lysine and 0.5 mM sample buffer), γ is equal to9.7. FIG. 14 shows two injection profiles for didansyl-lysine injectedfor 2 s with γ equal to 0.97 and 9.7. The injection profile with γ=0.97(the separation and sample buffers are both 5 mM) shows no stacking. Thesecond profile with γ=9.7 shows a modest enhancement of 3.5 for relativepeak heights over the injection with γ=0.97 Didansyl-lysine is an anion,and thus stacks at the rear boundary of the sample buffer plug. Inaddition to increasing the analyte concentration, the spatial extent ofthe plug is confined. The injection profile with γ=9.7 has a width athalf-height of 0.41 s, while the injection profile with γ=0.97 has awidth at half-height of 1.88 s. The electric field strength in the,separation channel 34D during the injection (injection field strength)is 95% of the electric field strength in the separation channel duringthe separation (separation field strength). These profiles are measuredwhile the separation field strength is applied. For an injection time of2 s, an injection plug width of 1.9 s is expected for γ=0.97.

[0129] The concentration enhancement due to stacking was evaluated forseveral sample plug lengths and relative conductivities of theseparation buffer and analyte. The enhancement due to stacking increaseswith increasing relative conductivities, γ.In Table 1, the enhancementis listed for g from 0.97 to 970. Although the enhancement is largestwhen γ=970, the separation efficiency suffers due to an electroosmoticpressure originating at the concentration boundary when the relativeconductivity is too large. A compromise between the stacking enhancementand separation efficiency must be reached and γ=10 has been found to beoptimal. For separations performed using stacked injections with γ=97and 970, didansyl-lysine and dansyl-isoleucine could not be resolved dueto a loss in efficiency. Also, because the injection process on themicrochip is computer controlled, and the column is not physicallytransported from vial to vial, the reproducibility of the stackedinjections is 2.1% rsd (percent relative standard deviation) for peakarea for 6 replicate analyses. For comparisor, the non-stacked, gatedinjection has a 1.4% rsd for peak area for 6 replicate analyses, and thepinched injection has a 0.75% rsd for peak area for 6 replicateanalyses. These correspond well to reported values for large-scale,commercial, automated capillary electrophoresis instruments. However,injections made on the microchip are ≈100 times smaller in volume, e.g.100 pL on the microchip versus 10 nL on a commercial instrument TABLE 1Variation of stacking enhancement with relative conductivity, γ. γConcentration Enhancement 0.97 1 9.7 6.5 97 11.5 970 13.8

[0130] Buffer streams of different conductivities can be accuratelycombined on microchips. Described herein is a simple stacking method,although snore elaborate stacking schemes can be employed by fabricatinga microchip with additional buffer reservoirs. In addition, the leadingand training electrolyte buffers can be selected to enhance (lie samplestacking, and ultimately, to lower the detection limits beyond thatdemonstrated here. It is also noted that much larger enhancements areexpected for inorganic (elemental) cations due to the combination offield amplified analyte injection and better matching of analyte andbuffer ion mobilities.

[0131] Regardless of whether sample stacking is used, the microchiplaboratory system 10D of FIG. 12 can be employed to achieveelectrophorectic separation of an analyte composed of rhodamine B andsulforhodamine. FIG. 15 are electropherograms at (a) 3.3 cm, (b) 9.9 cm,and (c) 16.5 can from the point of injection for rhodamine B (lessretained) and sulforhodamine (more retained). These were taken using thefollowing conditions: injection type was pinched, E_(inj)=500 V/cm,E_(run)=170 V/cm, buffer=50 mM sodium tetraborate at pH 9.2. To obtainelectropherograms in the conventional manner, single point detectionwith the helium-neon laser (green line) was used at different locationsdown the axis of the separation channel 34D.

[0132] An important measure of the utility of a separation system is thenumber of plates generated per unit time, as given by the formula

N/t=L(Ht)

[0133] where N is the number of theoretical plates, t is the separationtime, L is the length of the separation column, and 14 is the heightequivalent to a theoretical plate. The plate height, H, can be writtenas

H=A+B/u

[0134] where A is the sum of the contributions from the injection pluglength and the detector path length, B is equal to 2D_(m) where D_(m) isthe diffusion coefficient for the analyte in the buffer, and u is thelinear velocity of the analyte.

[0135] Combining the two equations above and substituting u=μE where μis the effective electrophoretic mobility of the analyte and E is theelectric field strength, the plates per unit time can be expressed as afunction of the electric field strength:

N/t=(μE)²/(AμE+B)

[0136] At low electric field strengths when axial diffusion is thedominant form of band dispersion, the term AμE is small relative to Band consequently, the number of plates per second increases with thesquare of the electric field strength.

[0137] As the electric field strength increases, the plate heightapproaches a constant value, and the plates per unit time increaseslinearly with the electric field strength because B is small relative toAμE. It is thus advantageous to have A as small as possible, a benefitof the pinched injection scheme.

[0138] The efficiency of the electrophorectic separation of rhodamine Band sulforhodamine at ten evenly spaced positions was monitored, eachconstituting a separate experiment. At 16.5 cm from the point ofinjection, the efficiencies of rhodamine B and sulforhodamine are 38,100and 29,000 plates, respectively. Efficiencies of this magnitude aresufficient for many separation applications. The linearity of the dataprovides information about the uniformity and quality of the channelalong its length. If a defect in the channel, e.g., a large pit, waspresent, a sharp decrease in the efficiency would result; however, nonewas detected. The efficiency data are plotted in FIG. 16 (conditions forFIG. 16 were the same as for FIG. 15).

[0139] A similar separation experiment was performed using the microchipanalyte injector 10B of FIG. 6. Because of the straight separationchannel 34B, the analyte injector 10B enables faster separations thanare possible using the serpentine separation channel 34D of thealternate analyte injector 10D shown in FIG. 12. In addition, theelectric field strengths used were higher (470 V/cm and 100 V/cm for thebuffer and separation channels 26B, 34B, respectively), which furtherincreased the speed of the separations.

[0140] One particular advantage to the planar microchip laboratorysystem 10B of the present invention is that with laser inducedfluorescence the poi it of detection can be placed anywhere along theseparation column. The electropherograms are detected at separationlengths of 0.9 mm, 1.6 mm and 11.1 mm from the injection intersection40B. The 1.6 mm and 11.1 mm separation lengths were used over a range ofelectric field strengths from 0.06 to 1.5 kV/cm, and the separations hadbaseline resolution over this range. At an electric field strength of1.5 kV/cm, the analytes, rhodamine B and fluorescein, are resolved inless than 150 ms for the 0.9 mm separation length, as shown in FIG.17(a), in less than 260 ms for the 1.6 mm separation length, as shown inFIG. 17(b), and in less than 1.6 seconds for the 11.1 mm separationlength, as shown in FIG. 17(c).

[0141] Due to the trapezoidal geometry of the channels, the uppercorners make it difficult to cut the sample plug away precisely when thepotentials are switched from the sample loading mode to the separationmode. Thus, the injection plug has a slight tail associated with it, andthis effect probably accounts for the tailing observed in the separatedpeaks.

[0142] In FIG. 18, the number of plates per second for the 1.6 mm and11.1 mm separation lengths are plotted versus the electric fieldstrength. The number of plates per second quickly becomes a linearfunction of the electric field strength, because the plate heightapproaches a constant value. The symbols in FIG. 18 represent theexperimental data collected for the two analytes at the 1.6 mm and 11.1mm separation lengths. The lines are calculated using thepreviously-stated equation and the coefficients are experimentallydetermined. A slight deviation is seen between the experimental data andthe calculated numbers, for rhodamine B at the 11.1 mm separationlength. This is primarily due to experimental error.

[0143] Electrochromatography

[0144] A problem with electrophoresis for general analysis is itsinability to separate uncharged species. All neutral species in aparticular sample will have zero electrophoretic mobility, and thus, thesame migration time. The microchip analyte injector 10D shown in FIG. 12can also be used to perform electrochromatography to separate non-ionicanalytes. To perform such electrochromatography, the surface of theseparation channel 34D was prepared by chemically bonding a reversephase coating to the walls of the separation channel after bonding thecover plate to) the substrate to enclose the channels. The separationchannel was treated with 1 M sodium hydroxide and then rinsed withwater. The separation channel was dried at 125° C. for 24 hours whilepurging with helium at a gauge pressure of approximately 50 kPa. A 25%(w/w) solution of chlorodimethyloctaldecylsilane (ODS, Aldrich) intoluene was loaded into the separation channel with an over pressure ofhelium at approximately 90 kPa. The ODS/toluene mixture was pumpedcontinuously into the column throughout the 18 hour reaction period at125° C. The channels are rinsed with toluene and then with acetonitrileto remove the unreacted ODS. The laboratory system 10D was used toperform electrochromatography on an analytes composed of coumarin 440(C440), coumarin 450 (C450) and coumarin 460 (C460; Exciton ChemicalCo., Inc.) at 10 μM for the direct fluorescent measurements of theseparations and 1 μM for the indirect fluorescent measurements of thevoid time. A sodium tetraborate buffer (10 mM, pH 9.2) with 25% (v/v)acetonitrile was the buffer.

[0145] The analyte injector 10D was operated under a pinched analyteloading mode and a separation (run) mode as described above with respectto FIG. 6. The analyte is loaded into the injection cross via a frontalchromatogram traveling from the analyte reservoir 16D to the analytewaste reservoir 18D, and once the front of the slowest analyte passesthrough the injection intersection 40D, the sample is ready to beanalyzed. To switch to the separation mode, the applied potentials arereconfigured, for instance by manually throwing a switch. Afterswitching the applied potentials, the primary flow path for theseparation is from the buffer reservoir 12D to the waste reservoir 20D.In order to inject a small analyte plug into the separation channel 34Dand to prevent bleeding of the excess analyte into the separationchannel, the analyte and the analyte waste reservoirs 16D, 18D aremaintained at 57% of the potential applied to the buffer reservoir 12D.This method of loading and injecting the sample is time-independent,non-biased and reproducible.

[0146] In FIG. 19, a chromatogram of the coumarins is shown for a linearvelocity of 0.65 mm/s. For C440, 11700 plates was observed whichcorresponds to 120 plates/s. The most retained component, C460, has anefficiency nearly an order of magnitude lower than for C440, which was1290 plates. The undulating background in the chromatograms is due tobackground fluorescence from the glass substrate and shows the powerinstability of the laser. This, however, did not hamper the quality ofthe separations or detection. These results compare quite well withconventional laboratory High Performance LC (HPLC) techniques in termsof plate numbers and exceed HPLC in speed by a factor of ten. Efficiencyis decreasing with retention faster than would be predicted by theory.This effect may be due to overloading of the monolayer stationary orkinetic effects due to the high speed of the separation.

[0147] Micellar Electrokinetic Capillary Chromatography

[0148] In the electrochromatography experiments discussed above withrespect to FIG. 19, sample components were separated by theirpartitioning interaction with a stationary phase coated on the channelwall. Another method of separating neutral analytes is micellarelectrokinetic capillary chromatography (MECC). MECC is an operationalmode of electrophoresis in which a surfactant such as sodiumdodecylsulfate (SDS) is added to the buffer in sufficient concentrationto form micelles in the buffer. In a typical experimental arrangement,the micelles move much more slowly toward the cathode than does thesurrounding buffer solution. The partitioning of solutes between themicelles and the surrounding buffer solution provides a separationmechanism similar to that of liquid chromatography.

[0149] The microchip laboratory 10D of FIG. 12 was used to perform on ananalyte composed of neutral dyes coumarin 440 (C440), coumarin 450(C450), and coumarin 460 (C460, Exciton Chemical Co., Inc.). Individualstork solutions of each dye were prepared in methanol, then diluted intothe analysis buffer before use. The concentration of each dye wasapproximately 50 μM unless indicated otherwise. The MECC buffer wascomposed of 10 mM sodium borate (pH 9.1), 50 mM SDS, and 10% (v/v)methanol. The methanol aids in solubilizing the coumarin dyes in theaqueous buffer system and also affects the partitioning of some of thedyes into the micelles. Due care must be used in working with coumarindyes as the chemical, physical, and toxicological properties of thesedyes have not been fully investigated.

[0150] The microchip laboratory system 10D was operated in the “pinchedinjection” mode described previously. The voltages applied to thereservoirs are set to either loading mode or a “run” (separation) mode.In the loading mode, a frontal chromatogram of the solution in theanalyte reservoir 16D is pumped electroosmotically through theintersection and into the analyte waste reservoir 18D. Voltages appliedto the buffer and waste reservoirs also cause weak flows into theintersection from the sides, and then into the analyte waste reservoir18D. The chip remains in this mode until the slowest moving component ofthe analyte has passed through the intersection 40D. At this point, theanalyte plug in the intersection is representative of the analytesolution, with no electrokinetic bias.

[0151] An injection is made by switching the chip to the “run” modewhich changes the voltages applied to the reservoirs such that buffernow flows from the buffer reservoir 120 through the intersection 40Dinto the separation channel 34D toward the waste reservoir 20D. The plugof analyte that was in the intersection 40D is swept into the separationchannel 34D. Proportionately lower voltages are applied to the analyteand analyte waste reservoirs 16D, 18D to cause a weak flow of bufferfrom the buffer reservoir 12D into these channels. These flows ensurethat the sample plug is cleanly “broken off” from the analyte stream,and that no excess analyte leaks into the separation channel during theanalysis.

[0152] The results of the MECC analysis of a mixture of C440, C450, andC460 are shown in FIG. 20. The peaks were identified by individualanalyses of each dye. The migration time stability of the first peak,C440, with changing methanol concentration was a strong indicator thatthis dye did not partition into the micelles to a significant extent.Therefore it was considered an electroosmotic flow marker with migrationtime t0. The last peak, C460, was assumed to be a marker for themicellar migration time, tm. Using these values of to and tin from thedata in FIG. 20, the calculated elution range, t0/tm, is 0.43. This adswell with a literature value of t0/tm=0.4 for a similar buffer system,and supports our assumption. These results compare well withconventional MECC performed in capillaries and also shows someadvantages over the electrochromatography experiment described above inthat efficiency is retained with retention ratio. Further advantages ofthis approach to separating neutral species is that no surfacemodification of the walls is necessary and that the stationary phase iscontinuously refreshed during experiments.

[0153] Inorganic Ion Analysis

[0154] Another laboratory analysis that can be performed on either thelaboratory system 10B of FIG. 6 or the laboratory system 10D of FIG. 12is inorganic ion analysis. Using the laboratory system 10B of FIG. 6,inorganic ion analysis was performed on metal ions completed with8-hydroxyquinoline-5-sulfonic acid (HQS) which are separated byelectrophoresis and detected with UV laser induced fluorescence. HQS hasbeen widely used as a ligand for optical determinations of metal ions.The optical properties and the solubility of HQS in aqueous media haverecently been used for detection of metal ions separated by ionchromatography and capillary electrophoresis. Because uncomplexed HQSdoes not fluoresce, excess ligand is added to the buffer to maintain thecomplexation equilibria during the separation without contributing alarge background signal. This benefits both the efficiency of theseparation and detectability of the sample. The compounds used for theexperiments are zinc sulfate, cadmium nitrate, and aluminum nitrate. Thebuffer is sodium phosphate (60 mM, pH 6.9) with8-hydroxyquinoline-5-sulfonic acid (20 mM for all experiments exceptFIG. 5; Sigma Chemical Co.). At least 50 mM sodium phosphate buffer isneeded to dissolve up to 20 mM HQS. The substrate 49B used was fusedquartz which provides greater visibility than glass substrates.

[0155] The floating or pinched analyte loading, as described previouslywith respect to FIG. 6, is used to transport the analyte to theinjection intersection 40B. With the floating sample loading, theinjected plug has no electrophoretic bias, but the volume of sample is afunction of the sample loading time. Because the sample loading time isinversely proportional to the field strength used, for high injunctionfield strengths a shorter injection time is used than for low injectionfield strengths. For example, for an injection field strength of 630V/cm FIG. 3a), the injection time is 12 s, and for an injection fieldstrength of 520 V/cm (FIG. 3b), the injection time is 14.5 s. Both thepinched and floating sample loading can be used with and withoutsuppression of the electroosmotic flow.

[0156] FIGS. 21(a) and 21(b) show the separation of three metal ionscomplexed with 8-hydroxyquinoline-5-sulfonic acid. All three complexeshave a net negative charge. With the electroosmotic flow minimized bythe covalent bonding of polyacrylamide to the channel walls, negativepotentials relative to ground are used to manipulate the complexesduring sample loading and separation. In FIGS. 21(a) and 21(b), theseparation channel field strength is 870 and 720 V/cm, respectively, andthe separation length is 16.5 mm. The volume of the injection plug is120 pL which corresponds to 16, 7, and 19 fmol injected for Zn, Cd, andAl, respectively, for FIG. 4 a. In FIG. 4b, 0.48, 0.23, and 0.59 fmol ofZn, Cd, and Al, respectively, are injected onto the separation column.The average reproducibility of the amounts injected is 1.6% rsd (percentrelative standard deviation) as measured by peak areas (6 replicateanalyses). The stability of the laser used to excite the complexes is≈1% rsd. The detection limits are in a range where useful analyses canbe performed.

[0157] Post-Separation Channel Reactor

[0158] An Iternate microchip laboratory system 10E is shown in FIG. 22.The five-port pattern of channels is disposed on a substrate 49E andwith a cover slip 49E′, as in the previously-described embodiments. Themicrochip laboratory system 10E embodiment was fabricated using standardphotolithographic, wet chemical etching, and bonding techniques. Aphotomask was fabricated by sputtering chrome (50 nm) onto a glass slideand ablating the channel design into the chrome film via a CAD/CAM laserablation system (Resonetics, Inc.). The channel design was thentransferred onto the substrates using a positive photoresist. Thechannels were etched into the substrate in a dilute Hf/Nh₄F bath. Toform the separation channel 34E, a coverplate was bonded to thesubstrate over the etched channels using a direct bonding technique. Thesurfaces were hydrolyzed in dilute NH₄OH/H₂O₂ solution, rinsed indeionized, filtered H₂, joined and then annealed at 500° C. Cylindricalglass reservoirs were affixed on the substrate using RTV silicone (madeby General Electric). Platinum electrode, provided electrical contactfrom the voltage controller 46E (Spellman CZE1000R) to the solutions inthe reservoirs.

[0159] The channel 26E is in one embodiment 2.7 mm in length from thefirst reservoir 12E to the intersection 40E, while the channel 30E is7.0 mm, and the third channel 32E is 6.7 mm. The separation channel 34Eis modified to be only 7.0 mm in length, due to the addition of areagent reservoir 22E which has a reagent channel 36E that connects tothe separation channel 34E at a mixing tee 44E. Thus, the length of theseparation channel 34E is measured from the intersection 40E to themixing tee 44E. The channel 56 extending from the mixing tee 44E to thewaste reservoir 20E is the reaction column or channel, and in theillustrated embodiment this channel is 10.8 mm in length. The length ofthe reagent channel 36E is 11.6 mm.

[0160] In a representative example, the FIG. 22 embodiment was used toseparate an analyte and the separation was monitored on-microchip viafluorescence using an argon ion laser (351.1 nm, 50 mW, Coherent Innova90) for excitation. The fluorescence signal was collected with aphotomultiplier tube (PMT, Oriel 77340) for point detection and a chargecoupled device (CCD, Princeton Instruments, Inc. TE/CCD-512TKM) forimaging a region of the microchip 90. The compounds used for testing theapparatus were rhodamine B (Exciton Chemical Co., Inc.) arginine,glycine, threonine and o-phthaldialdehyde (Sigma Chemical Co.). A sodiumtetraborate buffer (20 mM, pH 9.2) with 2% (v/v) methanol and 0.5% (v/v)β-mercaptoethanol was the buffer in all tests. The concentrations of theamino acid, OPA and rhodamine B solutions were 2 mM, 3.7 mM, and 50 μM,respectively. Several run conditions were utilized.

[0161] The schematic view in FIG. 23 demonstrates one example when 1 kVis applied to the entire system. With this voltage configuration, theelectric field strengths in the separation channel 34E (E_(scp)) and thereaction channel 36E (E_(rxa)) are 200 and 425 V/cm, respectively. Thisallows the combining of 1 part separation effluent with 1.125 partsreagent at the mixing tee 44E. An analyte introduction system such asthis, with or without post-column reaction, allows a very rapid cycletime for multiple analyses.

[0162] The electropherograms; (A) and (B) in FIG. 24 demonstrate theseparation of two pairs of amino acids. The voltage configuration is thesame as in FIG. 23, except the total applied voltage is 4 kV whichcorresponds to an electric field strength of 800 V/cm in the separationcolumn (E_(scp)) and 1,700 V/cm. in the reaction column (E_(rxa)). Theinjection times were 100 ms for the tests much correspond to estimatedinjection plug lengths of 384, 245, and 225 μm for arginine, glycine andthreonine, respectively. The injection volumes of 102, 65, and 60 pLcorrespond to 200, 130, and 120 fmol injected for arginine, glycine andthreonine, respectively. The point of detection is 6.5 mm downstreamfrom the mixing tee which gives a total column length of 13.5 mm for theseparation and reaction.

[0163] The reaction rates of the amino acids with the OPA are moderatelyfast, but not fast enough on the time scale of these experiments. Anincrease in the band distortion is observed because the mobilities ofthe derivatized compounds are different from the pure amino acids. Untilthe reaction is complete, the zones of unreacted and reacted amino acidwill move at different velocities causing a broadening of the analytezone. As evidenced in FIG. 24, glycine has the greatest discrepancy inelectrophoretic mobilities between the derivatized and un-derivatizedamino acid. To ensure that the excessive band broadening was not afunction of the retention time, threonine was also tested. Threonine hasa slightly longer retention time than the glycine; however thebroadening is not as extensive as for glycine.

[0164] To test the efficiency of the microchip in both the separationcolumn and the reaction column, a fluorescent laser dye, rhodamine B,was used as a probe. Efficiency measurements calculated from peak widthsat half height were made using the point detection scheme at distancesof 6 mm and 8 mm from the injection cross or 1 mm upstream and 1 mmdownstream from the mixing tee. This provided information on the effectsof the mixing of the two streams.

[0165] The electric field strengths in the reagent column and theseparation column were approximately equal, and the field strength inthe reaction column was twice that of the separation column. Thisconfiguration of the applied voltages allowed an approximately 1:1volume ratio of derivatizing reagent and effluent from the separationcolumn. As the field strengths increased, the degree of turbulence atthe mixing tee increased. At the separation distance of 6 mm (1 mmupstream from the mixing tee), the plate height as expected as theinverse of the linear velocity of the analyte. At the separationdistance of 8 mm (1 mm upstream from the mixing tee), the plate heightdata decreased as expected as the inverse of the velocity of theanalyze. At the separation distance of 8 mm (1 mm downstream from themixing tee), the plate height data decreases from 140 V/cm to 280 V/cmto 1400 V/cm. This behavior is abnormal and demonstrates a bandbroadening phenomena when two streams of equal volumes converge. Thegeometry of the mixing tee was not optimized to minimize this banddistortion. Above separation field strength of 840 V/cm, the systemstabilizes and again the plate height decreases with increasing linearvelocity. For E_(scp)=1400 V/cm, the ratio of the plate heights at the 8mm and 6 mm separation lengths is 1.22 which is not an unacceptable lossin efficiency for the separation.

[0166] The intensity of the fluorescence signal generated from thereaction of OPA with an amino acid was tested by continuously pumpingglycine down the separation channel to mix with the OPA at the mixingtee. The fluorescence signal from the OPA/amino acid reaction wascollected using a CCD as the product moved downstream from the mixingtee. Again the relative volume ratio of the OPA and glycine streams was1.125. OPA has a typical half-time of reaction with amino acids of 4 s.The average residence times of an analyte molecule in the window ofobservation are 4.68, 2.34, 1.17, and 0.58 s for the electric fieldstrengths in the reaction column (E_(rxm)) of 240, 480, 960, and 1920V/cm, respectively. The relative intensities of the fluorescencecorrespond qualitatively to this 4 s half-time of reaction. As the fieldstrength increases in the reaction channel, the slope and maximum of theintensity of the fluorescence shifts further downstream because theglycine and OPA are swept away from the mixing tee faster with higherfield strengths. Ideally, the observed fluorescence from the productwould have a step function of a response following the mixing of theseparation effluent and derivatizing reagent. However, the kinetics ofthe reaction and a finite rate of mixing dominated by diffusion preventthis from occurring.

[0167] The separation using the post-separation channel reactor employeda gated injection scheme in order to keep the analyte, buffer andreagent streams isolated as discussed above with respect to FIG. 3. Forthe post-separation channel reactions, the microchip was operated in acontinuous analyte loading/separation mode whereby the analyte wascontinuously pumped from the analyte reservoir 12E through the injectionintersection 40E toward the analyte waste reservoir 18E. Buffer wassimultaneously pumped from the buffer reservoir 16E toward the analytewaste and waste reservoirs 18E, 20E to deflect the analyte stream andprevent the analyte from migrating down the separation channel. Toinject a small aliquot of analyte, the potentials at the buffer andanalyte waste reservoirs 16E, 18E are simply floated for a short periodof time (≈100 ms) to allow the analyte to migrate down the separationchannel as an analyte injection plug. To break off the injection plug,the potentials at the buffer and analyte waste reservoirs 16E, 18E arereapplied.

[0168] The use of micromachined post-column reactors can improve thepower of post-separation channel reactions as an analytical tool byminimizing the volume of the extra-channel plumbing, especially betweenthe separation and reagent channels 34E, 36E. This microchip design(FIG. 22) was fabricated with modest lengths for the separation channel34E (7 mm) and reagent channel 36E (10.8 mm) which were more thansufficient for this demonstration. Longer separation channels can bemanufactured on a similar size microchip using a serpentine path toperform more difficult separations as discussed above with respect toFIG. 12. To decrease post-mixing tee band distortions, the ratio of thechannel dimensions between the separation channel 34E and reactionchannel 56 should be minimized so that the electric field strength inthe separation channel 34E is large, i.e. narrow channel, and in thereaction channel 56 is small, i.e., wide channel.

[0169] For capillary separation systems, the small detection volumes canlimit the number of detection schemes that can be used to extractinformation. Fluorescence detection remains one of the most sensitivedetection techniques for capillary electrophoresis. When incorporatingfluorescence detection into a system that does not have naturallyfluorescing analytes, derivatization of the analyte must occur eitherpre- or post-separation. When the fluorescent “tag” is short lived orthe separation is hindered by pre-separation derivatization, post-columnaddition of derivatizing reagent becomes the method of choice. A varietyof post-separation reactors have been demonstrated for capillaryelectrophoresis. However, the ability to construct a post-separationreactor with extremely low-volume connections to minimize banddistortion has been difficult. The present invention takes the approachof fabricating a microchip device for electrophoretic separations withan integrated post-separation reaction channel 56 in a single monolithicdevice enabling extremely low volume exchanges between individualchannel functions.

[0170] Pre-Separation Channel Reaction System

[0171] Instead of the post-separation channel reactor design shown inFIG. 22, the microchip laboratory system 10F shown in FIG. 25 includes apre-separation channel reactor. The pre-separation channel reactordesign shown in FIG. 25 is similar to that shown in FIG. 1, except thatthe first and second channels; 26F, 28F form a “goal-post” design withthe reaction chamber 42F rather than the “Y” design of FIG. 1. Thereaction chamber 42F was designed to be wider than the separationchannel 34F to give lower electric field strengths in the reactionchamber and thus longer residence times for the reagents. The reactionchamber is 96 μm wide at half-depth and 6.2 μm deep, and the separationchannel 34F is 31 μm wide at half-depth and 6.2 μm deep.

[0172] The microchip laboratory system 10F was used to perform on-linepre-separation channel reactions coupled with electrophoretic analysisof the reaction products. Here, the reactor is operated continuouslywith small aliquots introduced periodically into the separation channel34F using the gated dispenser discussed above with respect to FIG. 3.The operation of the microchip consists of three elements: thederivatization of amino acids with o-phthaldialdehyde (OPA), injectionof the sample onto the separation column, and the separation/detectionof the components of the reactor effluent. The compounds used for theexperiments were arginine (0.48 mM), glycine (0.58 mM), and OPA (5.1 mM;Sigma Chemical Co.). The buffer in all of the reservoirs was 20 mMsodium tetraborate with 2% (v/v) methanol and 0.5% (v/v)2-mercaptoethanol. 2-mercaptoethanol is added to the buffer as areducing agent for the derivatization reaction.

[0173] To implement the reaction the reservoirs 12F, 14F, 16F, 18F, and20F were simultaneously given controlled voltages of 0.5 HV, 0.5 HV, HV,0.2 HV, and ground, respectively. Thus configuration allowed the lowestpotential drop across the reaction chamber 42F (25 V/cm for 1.0 kVapplied to the microchip) and highest across the separation channel 34F(300 V/cm for 1.0 kV applied to the microchip) without significantbleeding of the product into the separation channel when using the gatedinjection scheme. The voltage divider used to establish the potentialsapplied to each of the reservoirs had a total resistance of 100 MΩ with10 MΩ divisiors. The analyte from the first reservoir 12F and thereagent from the second reservoir 14F are electroosmotically pumped intothe reaction chamber 42F with a volumetric ratio of 1:1.06. Therefore,the solutions from the analyte and reagent reservoirs 12F, 14F arediluted by a factor of ≈2. Buffer was simultaneously pumped byelectroosmosis from the buffer reservoir 16F toward the analyte wasteand waste reservoirs 18F, 20F. This buffer stream prevents the newlyformed product from bleeding into the separation channel 34F.

[0174] Preferably, a gated injection scheme, described above withrespect to FIG. 3, is used to inject effluent from the reaction chamber42F into the separation channel 34F. The potential at the bufferreservoir 16F is simply floated for a brief period of time (0.1 to 1.0s), and sample migrates into the separation channel. 34F. To break offthe injection plug, the potential at the buffer reservoir 16F isreapplied. The length of the injection plug is a function of both thetime of the injection and the electric field strength. With thisconfiguration of applied potentials, the reaction of the amino acidswith the OPA continuously generates fresh product to be analyzed.

[0175] A significant shortcoming of many capillary electrophoresisexperiments has been the poor reproducibility of the injections. Here,because the microchip injection process is computer controlled, and theinjection process involves the opening of a single high voltage switch,the injections can be accurately timed events. FIG. 26 shows thereproducibility of the amount injected (percent relative standarddeviation, % rsd, for the integrated areas of the peaks) for botharginine and glycine at injection field strengths of 0.6 and 1.2 kV/cmand injection times ranging from 0.1 to 1.0 s. For injection timesgreater than 0.3 s, the percent relative standard deviation is below1.8%. This is comparable to reported values for commercial, automatedcapillary electrophoresis instruments. However, injections made on themicrochip are ≈100 times smaller in volume, e.g. 100 pL on the microchipversus 10 nL on a commercial instrument. Part of this fluctuation is dueto the stability of the laser which is ≈0.6%. For injection times >0.3s, the error appears to be independent of the compound injected and theinjection field strength.

[0176]FIG. 27 shows the overlay of three electrophoretic separations ofarginine and glycine after on-microchip pre-column derivatization withOPA with a separation field strength of 1.8 kV/cm and a separationlength of 10 mm. The separation field strength is the electric fieldstrength in the separation channel 34F during the separation. The fieldstrength in the reaction chamber 42F is 150 V/cm. The reaction times forthe analytes is inversely related to their mobilities, e.g., forarginine the reaction time is 4.1 s and for glycine the reaction time is8.9 s. The volumes of the injected plugs were 150 and 71 pL for arginineand glycine, respectively, which correspond to 35 and 20 fmol of theamino acids injected onto the separation channel 34F. The gated injectorallows rapid sequential injections to be made. In this particular case,an analysis could be performed every 4 s. The observed electrophoreticmobilities for the compounds are determined by a linear fit to thevariation of the linear velocity with the separation field strength. Theslopes were 29.1 and 13.3 mm²/(kV−ás) for arginine and glycine,respectively. No evidence of Joule heating was observed as indicated bythe linearity of the velocity versus field strength data. A linear fitproduced correlation coefficients of 0.999 for arginine and 0.996 forglycine for separation field strengths from 0.2 to 2.0 kV/cm.

[0177] With increasing potentials applied to the microchip laboratorystem 10F, the field strengths in the reaction chamber 42F and separationchannel 34F increase. This leads to shorter residence times of thereactants in the reaction chamber and faster analysis times for theproducts. By varying the potentials applied to the microchip, thereaction kinetics can be studied. The variation in amount of productgenerated with reaction time is plotted in FIG. 28. The response is theintegrated area of the peak corrected for the residence time in thedetector observation window and photobleaching of the product. Theoffset between the data for the arginine and the glycine in FIG. 28 isdue primarily to the difference in the amounts injected, i.e. differentelectrophoretic mobilities, for the amino acids. A ten-fold excess ofOPA was used to obtain pseudo-first order reaction conditions. Theslopes of the lines fitted to the data correspond to the rates of thederivatization reaction. The slopes are 0.13 s⁻¹ for arginine and 0.11s⁻¹ for glycine corresponding to half-times of reaction of 5.1 and 6.2s, respectively. These half-times of reaction are comparable to the 4 spreviously reported for alanine. We have found no previously reporteddata for arginine or glycine.

[0178] These results show the potential power of integratedmicrofabricated systems for performing chemical procedures. The datapresented in FIG. 28 can be produced under computer control within fiveapproximately five minutes consuming on the order of 100 nL of reagents.These results are unprecedented in terms of automation speed and volumefor chemical reactions.

[0179] DNA Analysis

[0180] To demonstrate a useful biological analysis procedure, arestriction digestion and electrophoretic sizing experiment areperformed sequentially on the integrated biochemicalreactor/electrophoresis microchip system 10G shown in FIG. 29. Themicrochip laboratory system 10G is identical to the laboratory systemshown in FIG. 25 except that the separation channel 34G of thelaboratory system 10G follows a serpentine path. The sequence forplasmid pBR322 and the recognition sequence of the enzyme Hinf I areknown. After digestion, determination of the fragment distribution isperformed by separating the digestion products using electrophoresis ina sieving medium in the separation channel 34G. For these experiments,hydroxyethyl cellulose is used as the sieving medium. At a fixed pointdownstream in the separation channel 34G, migrating fragments areinterrogated using on-chip laser induced fluorescence with anintercalating dye, thiazole orange dimer (TOTO-1), as the fluorophore.

[0181] The reaction chamber 42G and separation channel 34G shown in FIG.29 are 1 and 67 mm long, respectively, having a width at half-depth of60 μm and a depth of 12 μm. In addition, the channel walls are coatedwith polyacrylamide to minimize electroosmotic flow and adsorption.Electropherograms are generated using single point detection laserinduced fluorescence detection. An argon ion laser (10 mW) is focused toa spot onto the chip using a lens (100 mm focal length) The fluorescencesignal is collected using a 21× objective lens (N.A.=0.42), followed byspatial filtering (0.6 mm diameter pinhole) and spectral filtering (560nm bandpass, 40 nm bandwidth), and measured using a photomultiplier tube(PMT). The data acquisition and voltage switching apparatus are computercontrolled. The reaction buffer is 10 mM Tris-acetate, 10 mM magnesiumacetate, and 50 mM potassium acetate. The reaction buffer is placed inthe DNA, enzyme and waste 1 reservoirs 12G, 14G, 18G shown in FIG. 29.The separation buffer is 9 mM Tris-borate with 0.2 mM EDTA and 1% (w/v)hydroxyethyl cellulose. The separation buffer is placed in the bufferand waste 2 reservoirs 16F, 20F. The concentrations of the plasmidpBR322 and enzyme Hinf I are 125 ng/μl and 4 units/μl, respectively. Thedigestions and separations are performed at room temperature (20° C.).

[0182] The DNA and enzyme are electrophoretically loaded into thereaction chamber 42G from their respective reservoirs 12G, 14G byapplication of proper electrical potentials. The relative potentials atthe DNA (12G), enzyme (14G), buffer (16G), waste 1 (18G), and waste 2(20G) reservoirs are 10%, 10% 0, 30%, and 100%, respectively. Due to theelectrophoretic mobility differences between the DNA and enzyme, theloading period is nude sufficiently long to reach equilibrium. Also, dueto the small volume of the reaction chamber 42G, 0.7 nL, rapiddiffusional mixing occurs. The electroosmotic flow is minimized by thecovalent immobilization of linear polyacrylamide, thus only anionsmigrate from the DNA and enzyme reservoirs 12G, 14G into the reactionchamber 42G with the potential distributions used. The reaction bufferwhich contains cations, required for the enzymatic digestions, e.g.Mg²⁺, is also placed in the waste 1 reservoir 18G This enables thecations to propagate into the reaction chamber countercurrent to the DNAand enzyme during the loading of the reaction chamber. The digestion isperformed statically by removing all electrical potentials after loadingthe reaction chamber 42G due to the relatively short transit time of theDNA through the reaction chamber.

[0183] Following the digestion period, the products are migrated intothe separation channel 34F for analysis by floating the voltages to thebuffer and waste 1 reservoirs 16F, 18F. The injection has a mobilitybias where the smaller fragments are injected in favor of the largerfragments. In these experiments the injection plug length for the75-base pair (bp) fragment is estimated to be 0.34 mm whereas for the1632-bp fragment only 0.22 mm. These plug lengths correspond to 34% and22% of the reaction chamber volume, respectively. The entire contents ofthe reaction clamber 42F cannot be analyzed under current separationconditions because the contribution of the injection plug length to theplate height would be overwhelming.

[0184] Following digestion and injection onto the separation channel34F, the fragments are resolved using 1.0% (w/v) hydroxyethyl celluloseas the sieving medium. FIG. 30 shows an electropherogram of therestriction fragments of the plasmid pBR322 following a 2 min digestionby the enzyme Hinf I. To enable efficient on-column staining of thedouble-stranded DNA after digestion but prior to interrogation, theintercalating dye, TOTO-1 (1 μM), is placed in the waste 2 reservoir 20Gonly and migrates countercurrent to the DNA. As expected the relativeintensity of the bands increases with increasing fragment size becausemore intercalation sites exist in the larger fragments. The unresolved220/221 and 507/511-bp fragments having higher intensities than adjacentsingle fragment peaks due to the band overlap. The reproducibility ofthe migration times and injection volumes are 0.55 and 3.1% relativestandard deviation (%rsd), respectively, for 5 replicate analyses.

[0185] This demonstration of a microchip laboratory system 10G thatperforms plasmid DNA restriction fragment analysis indicates thepossibility of automating and miniaturizing more sophisticatedbiochemical procedures This experiment represents the most sophisticatedintegrated microchip chemical analysis device demonstrated to date. Thedevice mixes a reagent with an analyte, incubates the analyte/reagentmixture, labels the products, and analyzes the products entirely undercomputer control while consuming 10,000 times less material than thetypical small volume laboratory procedure.

[0186] In general, the present invention can be used to mix differentfluids contained in different ports or reservoirs. This could be usedfor a liquid chromatography separation experiment followed bypost-column labeling reactions in which different chemical solutions ofa given volume are pumped into the primary separation channel and otherreagents or solutions can be injected or pumped into the stream atdifferent tunes to be mixed in precise and known concentrations. Toexecute this process, it is necessary to accurately control andmanipulate solutions in the various channels.

[0187] Pre-/Post-Separation Reactor System

[0188]FIG. 31 shows the same six port microchip laboratory system 10shown in FIG. 1, which could take advantage of this novel mixing scheme.Particular features attached to the different ports represent solventreservoirs. This laboratory system could potentially be used for aliquid chromatography separation experiment followed by post-columnlabeling reactions. In such an experiment, reservoirs 12 and 14 wouldcontain solvents to be used in a liquid chromatography solventprogramming type of separation, e.g., water and acetonitrile.

[0189] The channel 34 connected to the waste reservoir 20 and to the twochannels 26 and 28 connecting the analyte and solvent reservoir 12 and14 is the primary separation channel, i.e., where the liquidchromatography experiment would take place. The intersecting channels30, 32 connecting the buffer and analyte waste reservoirs 16 and 18 areused to make an injection into the liquid chromatography or separationchannel 34 as discussed above. Finally, reservoir 22 and its channel 36attaching to the separation channel 34 are used to add a reagent, whichis added in proportions to render the species separated in theseparation channel detectable.

[0190] To execute this process, it is necessary to accurately controland manipulate solutions in the various channels. The embodimentsdescribed above took very small volumes of solution (≈100 pl) fromreservoirs 12 and 40 and accurately injected them into the separationchannel 34. For these various scenarios, a given volume of solutionneeds to be transferred from one channel to another. For example,solvent programming for liquid chromatography or reagent addition forpost-column labeling reactions requires that streams of solutions bemixed in precise and known concentrations.

[0191] The mixing of various solvents in known proportions can be doneaccording to the present invention by controlling potentials whichultimately control electroosmotic flows as indicated in equation 1.According to equation 1 the electric field strength needs to be known todetermine the liner velocity of the solvent. In general, in these typesof fluidic manipulations a known potential or voltage is applied to agiven reservoir. The field strength can be calculated from the appliedvoltage and the characteristics of the channel. In addition, theresistance or conductance of the fluid in the channels must also beknown.

[0192] The resistance of a channel is given by equation 2 where R is theresistance, κ is the resistivity, L is the length of the channel, and Ais the cross-sectional area. $\begin{matrix}R_{i = \frac{\rho_{i}L_{i}}{A_{i}}} & (2)\end{matrix}$

[0193] Fluids are usually characterized by conductance which is just thereciprocal of the resistance as shown in equation 3. In equation 3, K isthe electrical conductance, ρ is the conductivity, A is thecross-sectional area, and L is the length as above. $\begin{matrix}K_{i = \frac{\kappa_{i}A_{i}}{L_{i}}} & (3)\end{matrix}$

[0194] Using ohms law and equations 2 and 3 we can write the fieldstrength in a given channel, i, in terms of the voltage drop across thatchannel divided by its length which is equal to the current, I_(i)through channel i times the resistivity of that channel divided by thecross-sectional area as shown in equation 4. $\begin{matrix}E_{i =_{L_{i}^{v_{i}} = {A_{i}^{l_{i}p_{i}} = {\kappa_{i}A_{i}^{l_{i}}}}}} & (4)\end{matrix}$

[0195] Thus, if the channel is both dimensionally and electricallycharacterized, the voltage drop across the channel or the currentthrough the channel can be used to determine the solvent velocity orflow rate through that channel as, expressed in equation 5. It is alsonoted that fluid flow depends on the zeta potential of the surface andthus on the chemical make-ups of the fluid and surface.

V_(i)∝I_(i)∝FLOW

[0196] Obviously the conductivity, κ, or the resistivity, ρ, will dependupon the characteristics of the solution which could vary from channelto channel. In many CE applications the characteristics of the bufferwill dominate the electrical characteristics of the fluid, and thus theconductance will be constant. In the case of liquid chromatography wheresolvent programming is performed, the electrical characteristics of thetwo mobile phases could differ considerably if a buffer is not used.During a solvent programming run where the mole fraction of the mixtureis changing, the conductivity of the ore may change in a nonlinearfashion but it will change monotonically from the conductivity of theone neat solvent to the other. The actual variation of the conductancewith mole fraction depends on the dissociation constant of the solventin addition to the conductivity of the individual ions.

[0197] As described above, the device shown schematically in FIG. 31could be used for performing gradient elution liquid chromatography withpost-column labeling for detection purposes, for example. FIG. 31(a),31(b), and 31(c) show the fluid flow requirements for carrying out thetasks involved in a liquid chromatography experiment as mentioned above.The arrows in the figures show the direction and relative magnitude ofthe flow in the channels. In FIG. 31(a), a volume of analyte from theanalyte reservoir 16 is loaded into the separation intersection 40. Toexecute a pinched injection it is necessary to transport the sample fromthe analyte reservoir 16 across the intersection to the analyte wastereservoir 18. In addition, to confine the lanalyte volume, material fromthe separation channel 34 and the solvent reservoirs 12,14 must flowtowards the intersection 40 as shown. The flow from the first reservoir12 is much larger than that from the second reservoir 14 because theseare the initial conditions for a gradient elution experiment. At thebeginning of the gradient elution experiment, it is desirable to preventthe reagent in the reagent reservoir 22 from entering the separationchannel 34. To prevent such reagent flow, a small flow of buffer fromthe waste reservoir 20 directed toward the reagent channel 36 isdesirable and this flow should be as near to zero as possible. After arepresentative analyte volume is presented at the injection intersection40, the separation can proceed.

[0198] In FIG. 31(b), the run (separation) mode is shown, solvents fromreservoirs 12 and 14 flow through the intersection 40 and down theseparation channel 34. In addition, the solvents flow towards reservoirs4 and 5 to make a clean injection of the analyte into the separationchannel 34. Appropriate flow of reagent from the reagent reservoir 22 isalso directed towards the separation channel. The initial condition asshown in FIG. 31(b) is with a large mole fraction of solvent 1 and asmall mole fraction of solvent 2. The voltages applied to the solventreservoirs 12, 14 are changed as a function of time so that theproportions of solvents 1 and 2 are changed from a dominance of solvent1 to mostly solvent 2. This is shown in FIG. 31(c). The latter monotonicchange in applied voltage effects the gradient elution liquidchromatography experiment. As the isolated components pass the reagentaddition channel 36, appropriate reaction can take place between thisreagent and the isolated material to form a detectable species.

[0199]FIG. 32 shows how the voltages to the various reservoirs arechanged for a hypothetical gradient elution experiment. The voltagesshown in this diagram only indicate relative magnitudes and not absolutevoltages. In the loading mode of operation, static voltages are appliedto the various reservoirs. Solvent flow from all reservoirs except thereagent reservoir 22 is towards the analyte waste reservoir 18. Thus,the analyte reservoir 18 is at the lowest potential and all the otherreservoirs are at higher potential. The potential at the reagentreservoir should be sufficiently below that of the waste reservoir 20 toprovide only a slight flow towards the reagent reservoir. The voltage atthe second solvent reservoir 14 should be sufficiently great inmagnitude to provide a net flow towards the injection intersection 40,but the flow should be a low magnitude.

[0200] In moving to the run (start) mode depicted in FIG. 31(b), thepotentials are readjusted as indicated in FIG. 32. The flow now is suchthat the solvent from the solvents reservoirs 12 and 14 is moving downthe separation channel 34 towards the waste reservoir 20. There is alsoa slight flow of solvent away from the injection intersection 40 towardsthe analyte and analyte waste reservoirs 16 and 18 and an appropriateflow of reagent from the reagent reservoir 22 into the separationchannel 34. The waste reservoir 20 now needs to be at the minimumpotential and the firs solvent reservoir 12 at the maximum potential. ARother potentials are adjusted to provide the fluid flow directions andmagnitudes as indicated in FIG. 31(b). Also, as shown in FIG. 32, thevoltages applied to the solvent reservoirs 12 and 14 are monotonicallychanged to move from the conditions of a large mole fraction of solvent1 to a large mole fraction of solvent 2.

[0201] At the end of the solvent programming run, the device is nowready to switch back to the inject condition to load another sample. Thevoltage variations shown in FIG. 32 are only to be illustrative of whatmight be done to provide the various fluid flows in FIGS. 31(a)-(c). Inan actual experiment some to the various voltages may well differ inrelative magnitude.

[0202] While advantageous embodiments have been chosen to illustrate theinvention, it will be understood by those skilled in the art thatvarious changes and modifications can be made therein without departingfrom the scope of the invention as defined in the appended claims.

What is claimed is:
 1. A method for separating component species in asample, comprising: a. providing a microfluidic device that includes abody having at least first, second, third and fourth channels disposedtherein, the body comprising a cover plate covering the first, second,third and fourth channels, wherein the first, second, third and fourthchannels communicate at a first intersection, the first channelconnecting at least a first sample source to the first intersection; b.transporting a sample material from the first sample source, through thefirst intersection and into the second channel by applying a firstvoltage difference between the first sample material source and thesecond channel to move sample material from the first channel, throughthe intersection and into the second channel, and simultaneouslyapplying a second voltage differences between the third channel and thefirst intersection and a third voltage difference between the fourthchannel and the first intersection, to direct movement of the samplematerial through the intersection into the second channel; and c.injecting an amount of sample material in the first intersection intothe third channel by applying a fourth voltage difference between thefirst intersection and the third channel, the component species of thesample material separating as the sample material is transported throughthe third channel.
 2. The method of claim 1, wherein the first channelcommunicates with the first intersection between the third and fourthchannels, and the third channel communicates with the first intersectionbetween the first and second channels, and wherein in the transportingstep, the second and third voltage differences move a material from thethird and fourth channels, respectively, into the second channel,pinching the sample material in the intersection.
 3. The method of claim2, wherein the injecting step further comprises removing the second andthird voltage differences concurrently with the step of applying thefourth voltage difference between the third and fourth channels throughthe first intersection, to move material in the intersection, into thethird channel.
 4. The method of claim 3, wherein the injecting stepfurther comprises applying a fifth voltage difference between the firstchannel and the first intersection, and a sixth voltage differencebetween the second channel, and the first intersection to move thesample material in the first and second channels away from theintersection.
 5. The method of claim 1, wherein the first channelcommunicates with the first intersection between the fourth channel andthe second channel and the second channel communicates with the firstintersection between the first channel and the third channel, andwherein in the transporting step, the second and third voltagedifferences applied in the transporting step move a material from thefourth channel into the third channel to gate movement of the samplematerial into the second channel.
 6. The method of claim 5, wherein theinjecting step further comprises removing the third voltage differenceconcurrently with the step of applying the fourth voltage differencebetween the first and third channels through the first intersection, tomove material in the first channel into the third channel.
 7. The methodof claim 1 further comprising the step of introducing a sieving mediuminto at least the third channel prior to the injecting step.
 8. Themethod of claim 7, wherein the sieving medium is introduced into thefirst, second, third and fourth channels.
 9. The method of claim 7,wherein the sieving medium is selected from cellulose and acrylamidepolymers.
 10. The method of claim 9, wherein the sieving medium isselected from hydroxyethylcellulose and polyacrylamide.
 11. The methodof claim 1, wherein the component species of the sample materialcomprise nucleic acids.
 12. The method of claim 11, wherein the nucleicacids comprise DNA.
 13. The method of claim 12, wherein the DNAcomprises restriction enzyme fragments of DNA.
 14. The method of claim11, wherein the nucleic acids comprise different size nucleic acids. 15.The method of claim 14, wherein the different size nucleic acids areprepared in a sequencing reaction.
 16. The method of claim 1, whereinthe component species of the sample material comprise proteins.
 17. Themethod of claim 16, wherein the sample material further comprises amicellar material.
 18. The method of claim 17, wherein the miscellarmaterial is sodium dodecyl sulfate.
 19. The method of claim 1 furthercomprising the step of detecting the separated component species. 20.The method of claim 19, wherein the third channel includes a detectionzone, and the detecting step comprises detecting the separated componentspecies in the third channel as the separated species are transportedpast the detection zone.
 21. The method of claim 19, wherein at least aportion of the component species comprise a fluorescent label, and thedetecting step comprises detecting fluorescence in the third channel.22. The method of claim 21, wherein the fluorescent label is afluorescein dye.
 23. The method of claim 21, wherein the label is arhodamine dye.
 24. The method of claim 21, wherein the component speciescomprise nucleic acids and the fluorescent label is an intercalatingdye.
 25. The method of claim 1, wherein the sample material comprisesionic species which are transported by electrophoresis.
 26. The methodof claim 1, wherein the sample material is transported through thefirst, second, third and fourth channels by electroosmosis.
 27. Themethod of claim 1, wherein the sample material is transported by acombination of electroosmosis and electrophoresis.
 28. The method ofclaim 1, wherein the first channel communicates with the intersectionbetween the third and fourth channels, and the third channelcommunicates with the intersection between the first and secondchannels, and wherein the transporting step comprises simultaneouslyelectrokinetically moving a material from the third and fourth channels,respectively, into the second channel, pinching the sample material inthe intersection.
 29. The method of claim 28, wherein the step ofelectrokinetically injecting a quantity of the sample material from theintersection into the third channel comprises concurrentlyelectrokinetically moving the sample material in the first and secondchannels away from the intersection.
 30. The method of claim 28 furthercomprising the step of detecting the separated component species in thethird channel.
 31. The method of claim 28, wherein the third channelincludes a detection zone, and the detecting step comprises detectingthe separated component species in the third channel as the separatedspecies are transported past the detection zone.
 32. The method of claim28, wherein at least a portion of the component species comprise afluorescent label, and the detecting step comprises detectingfluorescence in the third channel.
 33. The method of claim 32, whereinthe fluorescent label is a fluorescein dye.
 34. The method of claim 32,wherein the label is a rhodamine dye.
 35. The method of claim 32,wherein the component species comprise nucleic acids and the fluorescentlabel is an intercalating dye.
 36. A method for separating componentspecies in a sample, comprising: a. electrokinetically moving a samplematerial from a first covered microfluidic channel through anintersection of the first channel with second, third and fourth coveredmicrofluidic channels, and into the second channel, while simultaneouslyelectrokinetically moving material into the intersection from at leastone of the third and fourth channels to control movement of the samplematerial through the intersection; b. electrokinetically injecting aquantity of the sample material from the intersection into the thirdchannel; and c. electrokinetically separating the sample material intocomponent species in the third channel.
 37. The method of claim 36,wherein the sample material comprises ionic species which aretransported by electrophoresis.
 38. The method of claim 36, wherein thesample material is transported through the first, second, third andfourth channels by electroosmosis.
 39. The method of claim 36, whereinthe sample material is transported by a combination of electroosmosisand electrophoresis.